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Program in Stem Cell Biology and Regenerative Medicine, University of Florida, Gainesville, Florida, USA
Key Words. Green fluorescent protein • Multipotent astrocytic stem cell • Neurosphere • Neural stem cell • Subependymal zone • Transplantation
Correspondence: Gregory P. Marshall II, Ph.D., Program in Stem Cell Biology and Regenerative Medicine, University of Florida, 1600 SW Archer Road, Gainesville, Florida 32611, USA. Telephone: 352-392-0231; Fax: 352-392-0025; e-mail: gpm2{at}ufl.edu
Received on May 1, 2005;
accepted for publication on August 22, 2005.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The in vivo functional assay of BMT has led to a variety of methods for isolating cells from the bone marrow that possess HSC activity. HSCs can be enriched by selection for the cell surface expression profile Thy.1lo, Sca.1+, Lin– [3], their ability to exclude Hoechst dye [4], their size and density using counterflow centrifugal elutriation [5], and their expression of aldehyde dehydrogenase [6]. Although it is not yet known how these phenotypes relate to each other developmentally, all of the techniques enrich for a cell population that satisfies the in vivo functional definition of the HSC.
Currently, the HSC has not been shown to retain the aforementioned functional characteristics after in vitro expansion, a shortcoming attributed to an incomplete reconstruction of the bone marrow microenvironment by the selected culture conditions [7]. The resulting cultured hematopoietic cells display pluripotency and the ability to expand after passages in vitro yet lack the capacity to sustain long-term hematopoiesis in vivo. Cells of this phenotype have been deemed hematopoietic progenitor cells (HPCs) rather than true HSCs. This indicates that the in vitro conditions are not conducive to the retention of the self-renewal ability possessed by the HSC.
Neurogenesis in the adult mammalian brain is believed to be restricted to the subependymal zone (SEZ) [8, 9] and the sub-granule zone of the hippocampal dentate gyrus [10–12]. Neural stem cells (NSCs) in the hippocampus and SEZ produce migratory neuroblasts that differentiate into granule and periglomerular interneuons. SEZ neuoroblasts migrate to the olfactory bulb (OB) via a well defined glial pathway called the rostral migratory stream (RMS), eventually functionally integrating into the OB neural circuitry [9, 13–15]. Cells that are currently considered to be NSCs can be cultured to generate both a spherical aggregate of clonal cells known as a neurosphere [16–23] and a monolayer of multipotent astrocytic stem cells (MASCs) [24]. Both in vitro manifestations display the stem cell properties of multipotency and serial expansion in tissue culture. Both cultures can be induced to differentiate into neurons, astrocytes, and oligodendrocytes and are capable of increasing in number after repeated passages. Transplantation of green fluorescent protein (GFP)+ neurospheres or MASCs into regions of active neurogenesis results in donor-derived neuroblasts and neurons [23–28], with transplantation to the SEZ resulting in donor-derived migratory neurons and periglomerular interneurons in the RMS and OB of the host animal.
We set out to model the NSC system of transplantation and engraftment after the HSC system, for although neurospheres and MASCs possess stem cell characteristics in vitro, it is not known whether they are pluripotent and self-renewing in vivo. The RMS transplant model for neurospheres and MASCs provides an ideal system for the analysis of the in vivo capabilities of these neural "stem" cell cultures. If the transplanted cells are indeed stem cells, they should engraft and contribute to the SEZ, RMS, and OB for the life of the animal. It should also be possible to reisolate donor-derived neurospheres and MASCs, with the resultant cells capable of subsequent serial transplantation with similar evidence of engraftment.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Isolation and Culture of Neurospheres
Neurosphere cultures were generated from GFP+ transgenic neonatal mice (1–2 days postbirth) or transplanted adult C57BL/6 mice (see below) as described [19]. Briefly, animals were anesthetized with isofluorane, cervically dislocated, and decapitated. The brain was exposed, surgically removed, and then placed on an ice-cold sterile dissection board. A rectangular forebrain block containing the SEZ was obtained by removing the OB, cerebellum, hippocampus, lateral portions of the striatum, and lateral and dorsal cerebral cortex. The block was minced with a sterile scalpel and placed in ice-cold phosphate-buffered saline (PBS) containing antibiotic and antimycotic agents (Penicillin-Streptomycin, catalog no. 15140-122; Gibco, Carlsbad, CA, http://www.invitrogen.com, and Fungizone Antimycotic, catalog no. 15295-017; Gibco) for 10 minutes. Minced tissue was then centrifuged for 5 minutes at 1,100 rpm at 4°C, resuspended in 3 ml 0.25% Trypsin plus EDTA (catalog no. 25200-056; Gibco), and incubated at 37°C for 5 minutes. After the trypsin was neutralized by the addition of 1 ml of fetal bovine serum (FBS), the tissue was triturated into a single-cell suspension by pipetting through a series of descending-diameter fire-polished Pasteur pipettes. The cells were washed in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (catalog no. 11330-032; Gibco) at 1100 rpm for 5 minutes at 4°C and resuspended in neural growth medium (DMEM/F-12 with HEPES and L-glutamine (catalog no. 11330-032; Gibco), 5% FBS, N2 supplement (catalog no. 17502-048; Gibco), Glu-taMAX-1 supplement (catalog no. 35050-061; Gibco), epidermal growth factor (EGF; catalog no. 236-EG, 20 ng/ml; R&D Systems Inc., Minneapolis, http://www.rndsystems.com), and basic fibroblast growth factor (bFGF; catalog no. 03 116 999 001, 10 ng/ml; Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com). The cells were plated out in nonadhesive six-well plates (catalog no. 3471; Corning Costar, Acton, MA, http://www.corning.com) at a density of 1,000 cells per cm2. Cultures were supplemented with EGF and bFGF every second day.
Isolation and Culture of MASCs
Primary SEZ tissue was isolated from GFP+ transgenic neonatal mice (1–2 days postbirth) or transplanted adult C57BL/6 mice (see below) and dissociated to a single-cell suspension in the same manner as listed above. Cells were plated onto tissue culture flasks at high density in neural growth medium devoid of EGF and bFGF. Three days after the initial plating, nonadherent cells were removed, and fresh media was applied. Cultures were passaged once the resulting astrocytes had formed a confluent monolayer, and cultures were deemed suitable for transplantation once they had undergone three passages (necessary for removal of contaminating neurons in the cultures). The absence of neurons was verified by staining an aliquot of the culture with the pan-neuronal marker ß-III tubulin.
Transplantation of GFP+ Neurospheres into the Lateral Ventricles and the RMS of Adult C57BL/6 Mice
Passage-three GFP+ neurospheres were collected via trypsinization and resuspended in 1 ml of growth medium (see above). Once the cell number was calculated, cells were resuspended in a volume of growth media yielding 10,000 cells per µl. Recipient mice were anesthetized with Avertin (2-2-2 tribromoethanol, catalog no. T4 840-2; 2-methyl 2-butanol, catalog no. 240486; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), and the scalp was surgically exposed. Twenty thousand cells (2 µl) were stereotaxically injected into the lateral ventricle (LV) via a 5-µl Hamilton syringe (Hamilton, Reno, NV, http://www.hamiltoncompany.com) attached to a 28-gauge needle at the following coordinates: A-P, –0.2; M-L, –1.2; H-D, –2.5. RMS injections were stereotaxically injected at the following coordinates: A-P, 3.0; M-L, 0.8; H-D, 3.0. Transplanted animals were allowed to recover and were returned to general housing.
Transplantation of GFP+ MASCs and Neurospheres into the LVs of Neonatal C57BL/6 Mice
Passage-three GFP+ MASCs or neurospheres were collected via trypsinization and resuspended in 1 ml of growth medium (see above). Once the cell number was calculated, cells were resuspended in a volume of growth media yielding 75,000 to 100,000 cells per µl. Recipient C57BL/6 neonatal mice (day postbirth 1–3) were anesthetized by placement at –20°C for 5 minutes. Cells were transplanted in a volume of 1 µl via a 5-µl Hamilton syringe attached to a 28-gauge needle into the LV using the bregma skull suture as a reference point. After transplantation, neonatal mice were warmed to consciousness and returned to the mother’s cage prior to return to general housing.
Tissue Immunohistochemistry
Three weeks after transplantation, animals were given a lethal dose of the anesthetic Avertin before being perfused through the left ventricle with 4% paraformaldehyde (PFA) in PBS. After perfusion, the brain was removed and post-fixed overnight by immersion in 4% PFA at 4°C. Fixed brains were then serially sectioned through the sagittal plane at 40 µm using a Leica vibratome (model VT-1000-S; Leica Microsystems AG, Wetz lar, Germany, http://www.leica.com) equipped with a sapphire blade. Tissue was prepared for immunohistochemistry by blocking at room temperature for 1 hour in PBS containing 10% FBS and 0.01% Triton X-100. Primary antibodies were applied to the sections overnight with moderate agitation at 4°C.
Residual primary antibody was removed by three 5-minute washes (PBS plus 0.01% Triton X-100), and secondary antibodies were applied at room temperature for 50 minutes. Finally, sections were washed in PBS three times for 5 minutes, mounted on positively charged glass slides (Fisherbrand Superfrost/Plus, catalog no. 12-550-15; Fischer Scientific Co., Pittsburgh, https://www1.fishersci.com) and allowed to dry for 15 minutes at 37°C before being cover-slipped in Vectashield (H-1000; Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) mounting medium. Sections were analyzed and photographed by fluorescence microscopy using a Zeiss Axioplan 2 upright microscope (Carl Zeiss, Thornwood, NY, http://www.zeiss.com), Leica DMLB, or a Leica TCS SP2 AOBS spectral confocal microscope.
Selection of GFP+ MASCs by Puromycin Selection
MASC cultures derived from the SEZ of GFP+ and C57BL/6 neonatal and adult mice were prepared as described above. Once a confluent monolayer had been established, cells were passaged at 25% confluence with puromycin stock solution added to the neural growth medium at a final concentration of 2 µg/ml. Cells were exposed to puromycin in the neural growth medium for 9 days, with media replaced with fresh neural growth medium plus puromycin on the fifth day. In order for surviving cells to generate a secondary confluent monolayer, cells were collected by trypsinization and replated at highest density in puromycin-free neural growth medium. After generation of a secondary monolayer, the above selection protocol was repeated to ensure the removal of all puromycin-sensitive cells. Resultant cells were analyzed after removal of puromycin by phase-contrast microscopy.
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RESULTS |
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Neurogenesis in the neonatal (P 1–3) mouse brain is more active than observed in the adult [29], theoretically providing higher levels of extracellular cues for differentiation and engraftment of transplanted NSCs. Therefore, we also transplanted GFP+ neurospheres cultured from P 3 neonatal mice (75,000 cells, 1 µl) into the LV of P 1–3 C57BL/6 mice (n = 9) and allowed engraftment for 3 weeks. Immunohistochemical analysis of three transplanted animals indicated engraftment levels higher than those seen in the previous adult transplants, with donor-derived cells present in the SEZ, RMS, and OB (Fig. 1
). The morphology of the donor-derived cells in the OB (Fig. 1B) is similar to granule neurons, indicating that the transplanted neurospheres differentiated from an immature cell to that of a terminally differentiated neuron. This differentiation occurred presumably via a migratory neuroblast intermediate, given that donor-derived neuroblasts were observed in the RMS in the majority of all transplants.
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GFP+ MASCs Fail to Self-Renew In Vivo
In addition to the neurosphere, we tested another in vitro manifestation reported to exhibit NSC activity: the MASC. Capable of in vitro expansion and of generating multipotent neurospheres, the MASC is suggested to be a manifestation of the in vivo NSC. Although neurosphere cultures are the oldest in vitro neural progenitor culture, the protocol for the generation and dissociation of neurospheres results in low yield and extended culture conditions. Conversely, the MASC is relatively simple to culture and generates large cell numbers in a comparatively short time without the presence of mitogens required for neurosphere growth. To this end, GFP+ MASCs were used in RMS transplants to determine whether this more robust culture system retains in vivo NSC activity.
C57BL/6 neonatal mice (P 1–3, n = 50, seven independent experiments) were transplanted with GFP+ MASCs (100,000 cells per transplant in 1 µl) into the LV and harvested after 3 weeks. In each series of transplants, animals analyzed for engraftment exhibited levels similar to those seen in previous neurosphere transplants (Fig. 2). Donor-derived cells were present in the SEZ, RMS, and OB, with cells in the OB adopting a granule neuron morphology (Fig. 2B). Secondary GFP+ donor-derived MASC cultures derived from the forebrain of the remaining transplanted animals could not be established. All cultures resulted in recipient-derived MASCs with the same frequency and characteristics as age-matched control cultures.
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). Therefore, even with strong selective advantage, no secondary donor-derived MASCs can be recovered after in vivo transplantation.
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We transplanted a series of cohorts with both GFP+ neurospheres and GFP+ MASCs using direct RMS injections in adults and LV injections in neonates. The RMS system was analyzed for donor-derived GFP+ cell contribution as described above. Up to 1 month after transplant, migrating neuroblasts can be detected in the RMS (Fig. 4A, 4B). However, 2 months after transplant, no donor-derived cells were detected in the RMS in any transplant recipient (unpublished observations). But we were able to detect donor-derived GFP+ cells in the OB and surrounding the LV for up to 14 months after transplantation of GFP+ neurospheres into the LV (Fig. 4C). Analysis of sagittal sections from three animals revealed the existence of donor-derived cells in the cortex surrounding the needle tract, the SEZ, and OB in two of the three animals. These donor-derived cells appeared either as granule cells with extensive processes (Fig. 4D) or as cells with an astrocytic morphology (Fig. 4E). Of particular interest were the noticeable lack of migratory neuroblasts in the RMS of these animals and the lack of buildup of donor cells within the OB. The absence of these cells suggests that long-term contribution to the migratory neuroblast population and OB neurons was not provided by the transplanted cells. These data further demonstrate that cultured neurospheres and MASCs function as short-term neural progenitors in vivo but are incapable of reconstituting the complete neural regenerative pathway over time.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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The extensive investigation of hematopoiesis has produced a functional definition of a stem cell: a single cell that is capable of producing all cell types of a particular organ for the life of the animal via asymmetrical division in which both an exact duplicate of the stem cell and a lineage-committed progenitor daughter cell are generated. Additionally, the stem cell can reconstitute its native niche after transplantation, and this ability is retained after secondary transplantation, a phenomenon known as serial reconstitution [2]. Functional transplantation of a stem cell and its subsequent reconstitution of the niche is a vital requirement because the isolated cell can now be considered a useful tool for tissue repair. Candidate stem cells from other organs would need to meet the same criteria if they are to be classified as true stem cells.
Both donor neurospheres and MASCs engrafted into the recipient neural regenerative pathways upon transplantation into the LVs of adult mice, with donor-derived migratory neuroblasts observed in the RMS in the weeks immediately after transplantation. This would indicate that some level of engraftment occurred after transplantation, but it is not known whether the transplanted cells remained in primitive state and generated lineage-committed progeny while undergoing self-renewal (true engraftment) or whether the transplanted cells immediately entered into the RMS after transplantation (transient engraftment). Because neither the neurosphere nor the MASC displayed the ability to be reisolated after transplantation into proven regions of neurogenesis, it can be concluded that true engraftment failed to occur, with the transplanted cells capable only of transient engraftment. This limitation resulted in the inability of either cell types to survive subsequent serial transplantation (Table 1), further supporting the conclusion of transient engraftment by the transplanted cells.
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HSC engraftment into the bone marrow of myeloablated mice is evidenced by the robust generation of daughter cells representing both the myeloid and lymphoid lineages. The obvious evidence of functional engraftment by the transplanted HSC is that the myeloablated animal survives the previous lethal dose of radiation; the depleted bone marrow becomes repopulated by the transplanted HSC and its resultant progeny. Long-term (i.e., 3 months or longer) engraftment and hematopoietic contribution is a critical requirement in the definition of the HSC, because short-term, transient engraftment can be supplied by HPCs. The ability to survive serial transplantation while retaining the capacity to provide long-term bone marrow reconstitution fulfills the final requirement for classification as a true stem cell. Currently, the liver hepatocyte is the only ASC other than the HSC that has displayed the potential for serial, functional engraftment. In a liver repopulation assay, transplanted hepatocytes functionally contributed to the regenerating liver in a robust, serial fashion with contribution observed after the sixth transplantation [32].
Whether the inability for the transplanted neurospheres and MASCs to be reisolated is due to the cell population transplanted or the niche into which the cells were placed is not known. The failure observed in the adult model could be attributed to the decreased level of neurogenesis in the adult animal, resulting in fewer engrafted cells and subsequently fewer isolatable cells. However, because transplants into neonatal mice yielded the same results, the cause likely lies in the cells transplanted rather than in the niche itself. Both neurosphere and MASC cultures are heterogeneous in nature, with cells existing in varying stages of maturation. The cell types observed to be multipotent and proliferative in culture are potentially progenitor cells derived from a relatively small number of NSCs, implying that not every neurosphere or MASC in culture is an NSC. This concept has been proposed in a recent study in which cultures of neurospheres were determined to contain a heterogeneous population of neurospheres, with the population actually possessing the stem cell characteristics of NSCs being much smaller than originally thought [33].
It cannot be ruled out that comparing the SEZ niche with the bone marrow niche places an unfair onus upon the transplantability of cultured NSCs. The SEZ may not be as receptive to transplanted cells, nor may it promote robust, multilineage engraftment as is evidenced in HSC transplantation. It should be noted that injury to the bone marrow is a requirement for functional engraftment by transplanted HSCs, and a similar situation may exist with the SEZ. We have recently shown that mild injury to the adult brain by exposure to ionizing radiation allows for increased engraftment by transplanted MASCs [34]. Although no evidence of long-term engraftment was observed in the injured animals, it may be that some other form or level of injury is required for true engraftment of transplanted cells to occur in the SEZ.
These results may lead one to the following question: Do NSCs even exist in the adult brain? The observations of persistent neurogenesis in the adult brain in the aforementioned regions of neurogenesis would indicate that stem cell activity is present in those regions. Furthermore, we have observed that mild levels of radiation will temporarily deplete the levels of neurogenesis in the adult SVZ, but over time these levels return to near normal, further alluding to the presence of stem cell activity [34]. Assuming that an NSC population exists in the adult brain, it may be that the in vitro culture conditions necessary for the generation of neurospheres and MASCs render the NSC incapable of surviving subsequent transplantations. This phenomenon has been observed in the field of hematopoiesis, with resulting cultures of HSCs incapable of repopulating the ablated bone marrow in the adult mouse [7]. It is primarily for this reason that the HSC is manipulated as a primary tissue isolate rather than as a cultured entity. It may be that the NSC will require direct isolation in order for its properties to be completely understood.
An intriguing possibility is that the observed neurogenesis in the adult brain is not the result of an endogenous, isolatable NSC pool but rather the product of migratory ASCs that undergo a phenotypic shift upon integration into neurogenic regions of the brain and subsequently give rise to more lineage-committed neural progenitor cells.
Observations supporting the contribution of the HSC to adult neurogenesis are as numerous as they are conflicting. Donor-derived microglia and astrocytes [35] and neurons [36, 37] have been reported to exist in the brains of animals exposed to lethal irradiation and reconstituted with HSC transgenic for reporter proteins, whereas a recent investigation has argued strongly for the existence of nonfusion product, donor-derived neurons in the hippocampus of humans after sex-mismatched BMT [38]. Transplantation of bone marrow-derived cells into the brains of neonatal mice has recently been reported to result in the generation of NSCs derived from the transplanted cells, with these NSCs capable of contributing to neurogenesis in vivo as well as being isolatable as neurospheres [39]. It has also been suggested that 0.5% of astrocytes in the adult brain are generated from a bone marrow-derived cell [35], and it is generally accepted that the NSC of the SEZ (the type B cell) is a form of astrocyte [13, 40, 41]. With this in mind, it is not implausible to propose that adult neurogenesis is driven by an ASC residing in the bone marrow rather than by an endogenous, isolatable NSC.
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DISCLOSURES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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ACKNOWLEDGMENTS |
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REFERENCES |
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