Neural stem cells in the adult mammalian forebrain: A relatively quiescent subpopulation of subependymal cells

Neuron,

Vol. 13, 1071-1082,

November,

1994, Copyright

0 1994 by Cell Press

Neural Stem Cells in the Adult Mammalian Forebrain: A Relatively Quiescent Subpopulation of Subependymal Cells Cindi M. Morshead,* Brent A. Reynolds,+ Constance C. Craig,* Michael W. McBurney,* William A. Staines,* Dante Morassutti,* Samuel Weiss,+ and Derek van der Kooy*

of EGF (Reynolds and Weiss, 1992). These observations suggest that the EGF-responsive cells display characteristics of stem cells in vitro such as proliferation, self-renewal, and the production of differentiated

*University of Toronto Neurobiology Research Group Department of Anatomy and Cell Biology Toronto, Ontario M5S IA8 Canada +University of Calgary Departments of Anatomy and Pharmacology and Therapeutics Calgary, Alberta T2N 4Nl Canada *University of Ottawa Department of Medicine Ottawa, Ontario KIH 8M5 Canada

progeny (Hall and Watt, 1989; Potten and Loeffler, 1990; Reynolds and Weiss, 1993; and unpublished data). Theadult mammalian brain is mitoticallyquiescent, with the exception of the subependyma, which lines the lateral ventricles in the forebrain and extends rostrally toward the olfactory bulb (Smart and LeBlond, 1961; Sturrock and Smart, 1980; Morshead and van der Kooy, 1992). The subependyma is derived from the subventricular zone, which gives rise to forebrain neurons and glia during development (Smart, 1961). The subependyma persists throughout adult life as a heterogeneous population of undifferentiated cells, with up to one-third of the cells mitotically active (Morshead and van der Kooy, 1992). The proliferating population has a cell cycle time of approximately 12.7 hr in the mouse and undergoes steady-state division, with one cell dividing to give rise to two progeny, one of which undergoes cell death and the other of which continues to divide (Morshead and van der Kooy, 1992). Luskin (1993) has recently shown thatthe rostra1 subependyma contains a subpopulation of proliferating cells that migrate to the olfactory bulb in neonatal rat pups and differentiate into neurons. Some of the progeny of the constitutively proliferating subependymal cells in the adult brain may also become neurons or glia instead of undergoing cell death (Okano et al., 1993; Lois and Alvarez-Buylla, 1994). When subependymal cells in adult brains were labeled in vivo with tritiated thymidine (3H-thy), subsequently removed, and explant cultured in high levels of serum, some 3H-thy-positive cells differentiated into neurons and glia in vitro (Lois and Alvarez-Buylla, 1993). This work illustrates that, given the appropriate environment, the constitutively proliferating subependymal cells in the adult brain have the capacity to generate neurons and glia instead of undergoing cell death. Thus, subependymal cells in vivo possess some of the same stem cell characteristics shown by the EGFresponsivecells isolated invitro.Adult subependymal cells can proliferate, self-renew (in a steady-state mode of division), and terminally differentiate (with death being the ultimate terminal differentiation). We hypothesized that the subependyma in the adult forebrain is the in vivo source of stem cells isolated in vitro in the presence of EGF. We found that subependymal cells in vivo and EGF-responsive cells in vitro displayed common markers such as the EGF receptor (EGF-R) and nestin. Moreover, dissection of the subependyma proved to be necessary and sufficient for the isolation of stem cells in vitro. To test directly which cell population within the subependyma (the constitutively proliferating population

Summary Dissection of the subependyma from the lateral ventricle of the adult mouse forebrain is necessary and sufficient for the in vitro formation of clonally derived spheres of cells that exhibit stem cell properties such as selfmaintenance and the generation of a large number of progeny comprising the major cell types found in the central nervous system. Killing the constitutively proliferating cells of the subependyma in vivo has no effect on the number of stem cells isolated in vitro and induces a complete repopulation of the subependyma in vivo by relatively quiescent stem cells found within the subependyma. Depleting the relatively quiescent cell population within the subependyma in vivo results in a corresponding decrease in spheres formed in vitro and in the final number of constitutively proliferating cells in vivo, suggesting that a relatively quiescent subependymal cell is the in vivo source of neural stem cells. Introduction Neurogenesis occurs early in the developing mammalian brain (Raedler and Raedler, 1978; McConnell, 1988). With a few exceptions (Altman and Das, 1965; Kaplan and Hinds, 1977; Bayer et al., 1982; Luskin, 1993), neuronal production is complete within a few days of birth, and the adult mammalian brain lacks the ability to replace neurons lost to injury or disease. Recently, a population of cells has been isolated from the adult mouse brain that proliferate in response to epidermal growth factor (EGF) and produce cells that can differentiate into neurons and glia (Reynolds and Weiss, 1992). A subpopulation of the cells within the clonally derived spheres do not differentiate into neurons and glia, but instead continue to proliferate and form new spheres when subcloned in the presence

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Figure

1. Location

of the Subependyma

in the Adult

Schematic representation of a coronal section through brain of an adult mouse showing the lateral ventricles subependymal layer lining them (thick black lines).

Forebrain the foreand the

comprising up to 33% of the subependymal cells or the remaining relatively quiescent cells)contained the multipotential stem cell, we designed experiments based on studies of stem cells in the hemopoietic system. We took advantage of the finding that only mitotically active cells will incorporate 3H-thy into their DNA and undergo cell death following the administration of high doses of 3H-thy (Becker et al., 1965; Lajtha et al., 1969). Accordingly, we were able to kill the constitutively proliferating subependymal cells and spare the remaining subependymal cells. By assayingthe numberof EGF-responsivestem cells that generate spheres in vitro after killing the constitutively proliferating cells, we tested whether the constitutively proliferating cells or the relatively quiescent cells within the sub-ependyma were the source of in vitro stem cells. The kill experiments revealed that the source of multipotential stem cells isolated in vitro is not the constitutively proliferating population but a separate, relatively quiescent subependymal population within the adult brain. We suggest that the constitutively proliferating population, which is generated by the relatively quiescent stem cells, contains adult precursors to neurons and glia. Results ECF-R and Nestin Are Present in the Adult Subependyma To isolate clonally derived spheres from adult brains in vitro, dissociated cells are cultured in a serum-free defined medium containing EGF. In the absence of EGF, no proliferation or sphere formation is observed (Reynolds and Weiss, 1992), indicating that cellswhich give rise to spheres possess the EGF-R. We hypothesized that the in vivo source of stem cells in the adult brain was the subependymal region lining the lateral ventricles in the forebrain, since this region is mitotitally active in the adult (Smart and LeBlond, 1961; Sturrock and Smart, 1980; Morshead and van der Kooy, 1992) (Figure 1). ECF-R-immunoreactive cells were found lining the lateral ventricles in the rostra1 fore-

brain (Figure 2B). No EGF-R-immunoreactive cells were observed in the third or fourth ventricle subependyma. The EGF-R-immunoreactive cells in the forebrain were confined exclusively to the subependymal region of the lateral ventricles and were not observed in the surrounding brain tissue. However, only a subpopulation of the lateral ventricle subependymal cells were ECF-R immunoreactive (Figure 28). Injections of bromodeoxyuridine (BrdU) over 10 hr before sacrifice (which labels the entire population of constitutively proliferating subependymal cells [Morshead and van der Kooy, 19921) revealed that the majority of EGF-R-immunoreactive cell bodies were also BrdU immunoreactive. Nestin is an intermediate filament protein found in undifferentiated CNS cells (Cattaneo and McKay, 1990; Lendahl et al., 1990). Adult EGF-generated spheres in vitro are nestin immunoreactive (Reynolds and Weiss, 1992). In vivo, nestin-immunoreactive cells were seen throughout the subependyma of the lateral ventricles (Figure 2C) but were not present around the third ventricle. Similar to the EGF-R staining, the nestin staining was confined to the subependymal region and was not observed in the surrounding brain parenchyma. Although the nestin staining was very filamentous, making it difficult to quantitate the number of cells labeled, it was possible to discriminate subependymal cell bodies in some regions (Figures 2D and 2F). BrdU injections over IO hr revealed that the nestin-immunoreactive and BrdU-immunoreactive regions of the lateral ventricle subependymawere overlapping. At higher power there were examples of subependymal cells double-labeled for BrdU and nestin, as well as examples of BrdU-negative but nestin-positive cells (Figures 2D-2G). There appear to be examples of cells labeled only with BrdU within the subependyma; however, the filamentous nature of the nestin staining makes it difficult to conclude that these BrdU-labeled nuclei do not have nestin-immunoreactive processes extending from their cell bodies.

The Subependyma Is Necessary and Sufficient for Sphere Formation In Vitro To address whether the subependyma is the in vivo source of spheres isolated in vitro, different regions of the adult brain were dissected and cultured in the presence of EGF. The total numbers of spheres resulting from dissections of the hippocampus, cortex, striatum excluding the subependyma, and striatum includingthesubependymawerecountedafter8days in vitro(DIV; Figure3). Sphereformation resulted only when the subependyma lining the lateral ventricles was included in the dissection. Careful microdissection and culturing of the subependyma, excluding the surrounding striatum, also gave rise to spheres containing precursors for neurons and glia. These observations establish the subependyma as the in vivo source of cells that give rise to clonally derived spheres in vitro.

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The Number of Constitutively Proliferating Subependymal Ceils In Vivo Parallels the Number of Spheres Formed In Vitro Previous work has shown that the subependymal layer of the lateral ventricle is composed of a heterogeneous population of cells (Morshead and van der Kooy, 1992), with up to one-third of the cells constitutively proliferating with a cell cycle time of 12.7 hr. The remaining subependymal cells are either mitotically quiescent or have a much longer cell cycle time. The number of constitutively proliferating cells follows a rostrocaudal gradient such that the more rostra1 forebrain contains more proliferating cells (Smart, 1961; Altman, 1963). In the more caudal forebrain sections, there is adecrease in the number of proliferating cells, and at the level of the rostra1 portion of the third ventricle, no proliferating subependymal cells are seen. We asked whether there was a correlation between the number of constitutively proliferating cells found within the subependyma in different regions of the forebrain and the number of stem cells from the same regions that form spheres in vitro. The subependyma was divided into four regions: the olfactory bulbs, the rostra1 striatum extending from the front of the brain to the optic chiasm, the caudal striatum extending caudally from the optic chiasm and including tissue uptothe rostra1 fourthventricle,and thefourth ventricle extending into the medulla. There is a significant positive correlation between the numbers of spheres isolated in vitro from these regions and the numbers of constitutively proliferating cells seen in vivo in these same regions (r2 = 0.55; p< .Ol; Figure4). Unfortunately, the ratio of numbers of spheres in vitro to numbers of proliferating cells in vivo cannot be used to give an absolute estimate of how many constitutively proliferating cells arise from each stem cell in vivo. During the dissociation and culture procedure, some of the stem cells that give rise to spheres are lost, and thus the number of spheres formed is underestimated. Though the significant correlation indicates that the number of constitutively proliferating cells in vivo is a good relative predictor of the number of spheres isolated in vitro, these data do not address specifically whether the constitutively proliferating cells are the source of stem cells that form spheres in vitro. The Subependyma Is Repopulated after Depletion of the Constitutively Proliferating Cells We hypothesized that the constitutively proliferating subependymal cells were the stem cells in vivo that give rise to spheres in vitro. Therefore, depleting the normally proliferative cells in vivo should block sphere formation in vitro. Mice received three injections of high doses of 3H-thy once every 4 hr, a time less than the 5 phase (4.2 hr) of the constitutively proliferating subependymal population (Morshead and van der Kooy, 1992), to ensure that each cell entering S phase would incorporate the 3H-thy. High doses of 3H-thy kill cells by incorporating into the DNA during

S phase, resulting in cell death when the cells attempt to undergo mitosis, as well as directly killing the cells by intranuclear radiation (Becker et al., 1965; Lajtha et al., 1969). We sampled the number of proliferating subependymal cells after various sacrifice times (0.5, 1, 2, 4, 6, or 8 days post-kill) using BrdU. At 0.5 days post-kill, less than 1% of the control number of dividing subependymal cells were still proliferating (Figure 5A). No preferential localization of the few BrdUlabeled cells surrounding the ventricle at short survival times was observed. There was a progressive increaseovertimeinthenumberof proliferatingcells, and by 8 days post-kill, the number of proliferating subependymal cells was not significantly (t15 = 2.0; p > .05) different from control values. One possible mechanism for this repopulation is that the constitutively proliferating cells that took up 3H-thy did not die but simply stopped proliferating for a few days. We addressed this possibility by injecting animals with the same regimen of high dose 3H-thy on day 0 and sacrificing them on days 1 or 2 post-kill, before thecells hadachancetodiluteoutthe3H-thythrough possible cell divisions. A small number of 3H-thypositive cells were found within the subependyma at 1 day post-kill in sections processed autoradiographitally, indicating the survival of a few cells that took up3H-thy on day0. However, by2days the few heavily labeled cells seen on day 1 were no longer present, suggesting that they had died (data not shown). These data indicate that the repopulation of the subependyma post-kill cannot be accounted for by a failure to kill the constitutively proliferative subependymal cells. A second possible mechanism for repopulation after the 3H-thy kill involves the surviving cells of the constitutively proliferating population repopulating the subependyma. This mechanism would require minimally that the remaining subependymal cells changed their mode of proliferation from the normally observed steady-state mode (one progeny dies while the other survives and continues to proliferate; Morshead and van der Kooy, 1992) to a growth mode that increased the size of the proliferating population over time. A switch to growth mode within the surviving constitutively proliferating population would involve a change from asymmetric division (one of the progeny undergoes cell death) to symmetric division (both progeny survive and proliferate). However, given that less than 1% of the control number of subependymal cells are proliferating 0.5 days post-kill, not even exponential division of this population could produce the finding that 28% of control subependymal proliferation is seen 2 days post-kill. Even if both progeny of the proliferating 1% continue to divide exponentially, it is impossible for them to repopulate more than 8% of the constitutively proliferating population by day 2 post-kill (assuming three exponential divisions with a 12 hr cell cycle time). Furthermore, increasing the number of 3H-thy injections (five in total) and decreasing the interval between injections

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g 600 ab e “0 400 1 200

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Figure 3. Brain Dissections Reveal the Subependyma Vivo Source of Clonally Derived Spheres In Vitro

Is the In

The number of spheres formed after 8 DIV from separate dissections of the hippocampus, cortex, striatum excluding the subependyma, and striatum including the subependyma. Only dissections that included the subependyma resulted in sphere formation in vitro. Data represent means + SEM from four independent culture preparations; brains from two adult mice were pooled in each of the four independent cultures.

(one injection every 3 hr) did not change signficantly the repopulation curve observed in Figure 5A. This observation also suggests it is unlikely that the repopulation of the subependyma was a result of missing some of the constitutively proliferating cells with the series of 3H-thy kill injections. Relatively Quiescent Stem Cells Are Recruited to Repopulate the Proliferating Subependymal Population An alternative mechanism for repopulation of the dividing subependymal population is recruitment of a stem cell into S phase within the lateral ventricle subependyma. The stem cell must be mitotically quiescent at the time of the initial kill (to avoid uptake of 3H-thy and subsequent cell death) and then be recruited into S phase as a result of the death of the constitutively proliferating subependymal cells. We tested this recruitment hypothesis by utilizing the fact that, once a stem cell became mitotically active, it

Figure

2. Characterization

of Subependymal

Cells

0

Figure 4. Regional Analysis Reveals a Significant lation between the Number of Spheres In Vitro of Constitutively Proliferating Cells In Vivo

Positive Correand the Number

The number of spheres isolated from different subependymal regions after 8 DIV in the presence of ECF compared with the total number of constitutively proliferating subependymal cells in each of the same regions after 10 hrof BrdU injections. There is a significant positive correlation between the number of spheres isolated per region and the number of constitutively proliferating cells per region. Data represent means + SEM per animal.

would incorporate the high doses of 3H-thy and be killed (Becker et al., 1965). Animals received a second series bf 3H-thy injections on day 2 or 4 after the first kill on day 0, and the proliferating population was sampled 8 days later (days 10 and 12, respectively; Figure 58). When the second kill was done on day 2, only52% * 7% of thecontrol proliferating population was present on day 10 (8 days later), a time that is sufficient for the repopulation of proliferating subependymal cells after a single kill. This depletion was significantly (tg = 4.5; p < .05) different from control values and was maintained at day 31 post-kill, when 68% * 8% of the control proliferating population was observed, a value not significantly (t, = 1.7; p > .05) different from day 10 post-kill, yet still significantly (tq3 = 6.1; p < .05) different from controls. These find-

In Vivo

(A) Fluorescent photomicrograph of a coronal section through the subependyma surrounding the lateral ventricle in the adult forebrain from a mouse that received 10 hr of BrdU injections to label the entire constitutively proliferating population. The subependyma is heterogeneous, with a subpopulation of cells proliferating (BrdU-immunoreactive, arrows). (B) Fluorescent photomicrograph of a coronal section through the dorsolateral corner of the lateral ventricle showing ECF-Rimmunoreactive cells (arrows) within the subependyma. A subpopulation of cells within the subependyma are ECF-R immunoreactive. (C) Fluorescent photomicrograph of nestin staining in the subependyma of the dorsolateral corner of the lateral ventricle. The nestin staining is very filamentous, making it difficult to discern individual cell bodies. (D and E) Higher magnification photomicrographs of the dorsolateral corner (where up to 33% of the subependymal cells are constitutively proliferating) showing nestin-positive cell bodies (D) in one region within the subependyma and the same section doublelabeled forBrdU(E).Someofthesubependymalcellsaredoublelabeledforboth nestinandBrdU (arrows),whereasothernestin-immunoreactive cells are not BrdU labeled (arrowheads) and hence are not part of the constitutively proliferating population. (F and C) Arrows point to nestin-positive cells (F) in the ventral tip of the lateral ventricle that are double labeled for BrdU (G). The arrowhead points to an example of a nestin-positive cell (F) showing an empty nucleus that is not double labeled with BrdU (G). cc, corpus callosum; str, striatum; Iv, lateral ventricle.

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Figure 5. Repopulation after Depletion of the Constitutively Proliferating Subependymal Cells Reveals the Presenceof a Neural Stem Cell In Vivo

0

1

2 Time

4

in days

Time

after

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6 3H-thymidine

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days 0 and 2 kills

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days 0 and 4 kills

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(A) The number of BrdU-labeled proliferating subependymai cells at various survival times after a series of high doses of ?H-thy on day 0 (single kill) is expressed as a percentage of the number in control animais (loo%), which received BrdU only. (B) The number of BrdU-labeled proliferating subependymal cells at varous survival times after a series of 3H-thy injections on day 0 and a second series on day 2 or 4 (double kill). The continuous line joins points that were measured. The dotted lines join two points where at least one of the points was predicted (but not measured) based on the number of cells still proliferating 0.5 days after a kill (A). Data represent means + SEM of at least four mice for each time point, and the number of BrdU-labeled cells was averaged from six to eight sections per animal.

k!!i

ings suggest that, on day 2 post-kill, as many as 50% of the stem cells that repopulate the subependyma were mitotically active, incorporated 3H-thy into their DNA, and were killed. When the second kill was done on day4,84% i 4% of the control proliferating population was seen 8 days later (day 12), suggesting that by 4 days after the initial kill, most of the stem cells (which were recruited into the proliferative modeover the first few days) had become quiescent again and hence were not killed by the high doses of 3H-thy given on day 4. However, the finding that, by day 12 in the days 0 and 4 kill group, the proliferating population was still significantly (tl* = 4.8; p < .05) below control values suggests that a few (up to 15%) of the recruited stem cells still may have been proliferating on day 4. The double-kill data suggests a time window for the recruitment of relatively quiescent stem cells intothe proliferative mode afterthe initial depletion of the constitutively proliferating subependymal cells. The majority of stem cells enter the cell cycle over the first few days after the initial kill, and by day 4 post-kill, they are no longer mitotically active.

The Recruited Stem Cells In Vivo Are the Source of Spheres tn Vitro The double-kill experiments suggest the presence of a stem cell in the subependyma that is able to repopulate the lost constitutively proliferating population. Given the stem cell characteristics of the in vitro EGFresponsive cell (which is isolated from the subependyma), we asked whether the constitutively proliferating population or the relatively quiescent population was the in vivo source of EGF-generated spheres. We tested this hypothesis by counting the number of clonally derived spheres in vitro after each of the kill paradigms established in vivo. This provided a direct link from in vivo assessment of the number of proliferating subependymal cells to the in vitro isolation of stem cells. The number of proliferating cells was assessed in vivo 8 days after the last series of 3H-thy injections in each group (Figure 6A). In vitro, the mice were sacrificed 16-20 hr after the last series of 3H-thy injections, and the number of spheres isolated was counted after 8 DIV (Figure 6B). Animals that received a single kill with 3H-thy on day 0 (producing a greater

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Number (percent 0 20 8

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Figure 7. Model of the Lineage Stem Cell and the Constitutively 3H-thy

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Figure 6. The Relatively Quiescent Stem Cell Recruited Kill In Vivo Is the Source of Neural Stem Cells Isolated

after the In Vitro

(A) The number of proliferating subependymal cells in viva after single or double 3H-thy kills expressed as a percentage of the controls, 8 days following the last series of 3H-thy injections. These data were taken from the single- and double-kill in vivo experiments in Figure 5. (6) The number of spheres formed per brain expressed as a percentage of the control values, after single or double 3H-thy kills in vivo and culture for 8 days (starting 16-20 hr after the last series of Wthy injections). Data represent means + SEM for six mice per group.

than 99% loss of constitutively dymal cells 0.5 days post-kill any significant depletion isolated in vitro compared trol groups that received spheres [single kill] versus

proliferating subepen[Figure 5AJ) did not show in the number of spheres with the 100% value of consaline injections (66 f 9 65 * 7 spheres [control];

ts = 0.00; p > .05). This group reveals that the constitutively proliferating subependymal cells are not the source of stem cells that form spheres in vitro. When a second kill was done on day 2, there was a 50% reduction in the number of spheres isolated (33 k 5 [double kill]) compared with controls. This 50% reduction closely mirrors the 48% reduction in the proliferating subependymal cells in vivo after the second kill on day 2. We suggest both the in vivo and in vitro reductions reflect the killing of stem cells that had been recruited into the proliferative mode at the time

Relationship between the Neural Proliferating Cell Populations

Relatively quiescent stem cells (S) within the subependyma are multipotent and capable of self-renewal and give rise to clonally derived spheres in vitro. The progeny of the stem cells are the constitutively proliferating subependymal cells (C), which arise via an intermediary, the transit-amplifying cell 0. (X) marks cells destined to undergo cell death in vivo, although a subpopulation of the constitutively proliferating subependymal cells are the precursors for neurons and/or glia in vitro and serve as a source of new neurons for the olfactory bulb in adult mice in viva.

of the second series of 3H-thy injections, and that the recruited stem cell in vivo is the source of stem cells that give rise to multipotential spheres isolated in culture. To control for the total dose of 3H-thy administered, a group of animals received a double dose of 3H-thy on day 0. The total number of spheres (72 + 2; n = 6) formed in animals that received a double 3H-thy dose on day 0 was not significantly different from that of controls (ts = 1.0; p > .05). The final group of mice that received a second kill on day 4 provide a further test of the hypothesis that the recruited stem cell in vivo is the source of multipotential stem cells in vitro. By day4 in vivo, most of the stem cells responsible for repopulating the su bependyma are no longer mitotically active and hence are not killed by the second series of 3H-thy injections. As a result, the number of proliferating subependymal cells in vivo approached control values by day 12 (i.e., 8 days later). The sphere assay revealed no significant (ts = 0.8; p > .05) loss compared with controls in the number of spheres formed in vitro after a second kill on day 4 (56 f 9 spheres). This finding is consistent with a return to quiescence by the stem cell population, and thus protection from the second 3H-thy kill. The direct correlations between the in vivo proliferation assays and the in vitro stem cell assays reveal that a relatively quiescent cell within the subependyma (and not the

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Discussion The first major conclusion of this study is that the adult subependyma of the forebrain lateral ventricles is the in vivo source of a clonal neural stem cell that proliferates in vivo to generate constitutively proliferating subependymal cells and that proliferates in vitro to form spheres containing cells that can differentiate into neurons and glia. The support for this conclusion comes from the presence of nestin and EGF-R expression in both the adult forebrain subependyma and in spheres in vitro. More importantly, only dissection and culture of cells from the adult subependyma (and from no other adult forebrain area) will result in sphere formation. Thus, the adult subependyma is a necessary and sufficient source of neural stem cells. The second major conclusion is that the in vivo source of neural stem cells that produce spheres in vitro is not the constitutively proliferating su bependymal population but rather a relatively quiescent stem cell within the adult subependyma. Using high doses of 3H-thy to kill the constitutively proliferating subependymal cells in vivo has no effect on the capacity of cells from the subependyma to generate spheres after culture for 8 days in EGF. Moreover, the constitutively proliferating population can be entirely repopulated in vivo 8 days after a 3H-thy kill that reduces the constitutively proliferating population to less than 1% ofcontrol proliferation levels. Weconcludethat a relatively quiescent subependymal stem cell can both repopulate the constitutively proliferating population in vivo and serve as the source for sphere-forming stem cells in vitro (Figure 7). The relatively quiescent stem cell must be closely associated with the constitutively proliferating subependymal population in vivo because of the positive correlation between the absolute number of constitutively proliferating subependymal cells at different rostrocaudal forebrain levels in vivo and the numbers of spheres generated from tissue from each of these same levels in vitro. Direct evidence for the conclusion that the relatively quiescent subependymal population in vivo is the source of the in vitro stem cell comes from the double-kill experiments, which produced a long-term depletion of the constitutively proliferating subependymal population in vivo. This depletion results from killing off the relatively quiescent stem cells with high doses of 3H-thy after they have been recruited to proliferate in vivo following the first kill. Most important, there is a 48% depletion of constitutively proliferating subependymal cells after 8 days in vivo following the double-kill paradigm, which is matched by a 50% depletion in the numberof clonallyderived spheres generated in vitro following the same double-kill paradigm in vivo.

Although high dose 3H-thy kills on days 0 and 2 reduced by 50% both the number of proliferating subependymal cells in vivo (8 days post-kill) and the number of stem cell-generated spheres in vitro, similar kills on days 0 and 4 produced no such long-term losses in vivo or in vitro. This reveals the time course of the recruitment of stem cells into the proliferative mode. Over the first few days post-kill, stem cells pro.. liferate to replenish the constitutively proliferating population, and by day 4, the stem cell is no longer mitotically active. This time window for the recruitment of stem cells is similiar to that observed in the hemopoietic system. Harrison and Lerner (1991) found that there was little or no effect of a single treatment with the drug 5-fluorouracil on the primitive hemopoietic stem cells responsible for long-term repopulation of the mouse lymphoid and myeloid lineages. Like3H-thy, 5-fluorouracil targets rapidlyproliferating cells because incorporation of the nucelotide analog into DNA during the S phase of the cell cycle results in cell death (Chadwick and Rogers, 1972). However, when a second dose of 5-fluorouracil was administered 3-5 days after the initial dose, there was a significant loss in the repopulation capacity of the hemopoietic system. If the second dose was administered 1 or 8 days after the initial dose, there was no effect on repopulation. These findings suggest that the primitive hemopoietic stem cell normally cycles too slowly for a single dose of S-fluorouracii to be effective in killing a significant portion of the population. However, these stem cells are recruited into the cell cycle between 3 and 5 days after the initial treatment and become sensitive to the second dose. The 3-5 day time window in the hemopoietic system is roughly comparable to the early time frame (I-4 days) for neural stem cell recruitment observed in the subependyma. The kill experiments indicate that the adult subependyma contains a population of stem cells that areless mitoticallyactivethan theconstitutivelyproliferating population under normal conditions. Though the quiescent stem cells can be induced to proliferate as a result of massive cell death within the subependyma, this does not speak to the normal kinetics of these stem cells in vivo. When constitutively proliferating cells within the subependyma were labeled using a replication-deficient recombinant retrovirus injected into the lateral ventricle, the number of clones per brain decreased over time (Craig et al., 1994). By28 days post-retroviral infection, no retrovirally labeled cells were seen within the subependyma. Polymerase chain reaction analysis established that the subependymal cells had not simply turned off expression of the marker gene. This suggests that the constitutively proliferating population of cells has a finite life span of up to 28 days in the subependyma. Therefore, to maintain the size of the adult subependyma, these constitutively proliferating cells must be replaced. Since the stem cells within the subependyma are the presumed sourcefor repopulation,these datasuggest

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in the Adult

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that the stem cell would be relatively quiescent (with a long cell cycle time of up to 28 days). In support of this hypothesis, we have found that mice given 10 hr of BrdU injections (sufficient to label the entire constitutively proliferating population [Morshead and van der Kooy, 19921) and sacrificed after 28 days contain a population of less than 1% of the total subependymal population that remain BrdU-labeled (unpublished data). Together, the polymerase chain reaction and the long-term BrdU labeling both lend support to the finding that the subependyma contains a population of relatively quiescent stem cells. Since retrovirally labeled clones of constitutively proliferating adult subependymal cells are not seen in the subependyma for more than 28 days (Craig et al., 1994), the stem cell within the subependyma must proliferate occasionally and replace the constitutively proliferating cells in order to maintain the size of the forebrain subependyma in adult brains.Thestem cells may repopulate the subependyma in a stochastic manner, such that individual stem cells have a characteristic baseline probability of contributing to the replacement of subependymal cells. There is some evidence suggesting such a stochastic mode of repopulation in hemopoiesis (Korn et al., 1973; Spangrude et al., 1991). However, regulation of the proliferation of subependymal cells is required as well, since massive cell death within the constitutively proliferating population can increase dramatically the fraction of stem cells contributing to repopulation of the subependyma (50% of the stem cells are proliferating actively 2 days after depletion of the constitutively proliferating subependymal cells). Under these circumstances, stem cells may be induced to proliferate by the release of mitogens (Streit and Kreutzberg, 1988; Kiefer et al., 1993; Wen et al., 1994) from the dying subependymal cells or by the loss of cell-cell contact within the subependyma. It is also possible that the proliferation of stem cells within the subependyma is tightly regulated even under baseline conditions. For instance, if the ratio of subependymal stem cells to constitutively proliferating cells is l:l, then the death of a single constitutively proliferating cell maycausetheproliferationofasinglestemcelI,which will undergo an asymmetric division resulting in selfrenewal of the stem cell and aconstitutively proliferating cell. Alternatively, a single stem cell may be induced to proliferate only in response to the loss of several constitutively proliferating cells. In this scenario, the stem cell would proliferate to self-renew, and the other progeny would continue to proliferate in a growth mode to replace the lost subependymal cells. This scenario introducesathird type of proliferative cell, the dividing transit cell (Potten and Loeffler, 1990). This cell population amplifies each stem cell division and would be a transient intermediary between the stem cell and the constitutively proliferating cell. Both of these possibilities suggest a nonstochastic manner of proliferation and require a degree

of regulation within the system. The repopulation curve over 8 days after a single kill appears linear, which suggests that the repopulation occurs partially by way of a transit cell population. The double-kill experiment suggests that most of the subependymal stem cell recruitment is over by day 4 post-kill. If the stem cells were to give rise directly to the constitutively proliferating cells, the slope of subependymal repopulation after a single kill should be steep over the first 1-3 days post-kill and flatten out afterwards. The formation of spheres in vitro also suggests the presenceofa transit cell population. If thedirect progenyof the stem cells were the constitutively proliferating cells that proliferate in a steady-state mode of division, then the rapid clonal expansion from a stem cell into a multicellular sphere would not be possible. Figure 7 illustrates a proposed model for the lineage relationship between the relatively quiescent stem cell and the constitutively proliferating population. The model indicates that the stem cell gives rise to the constitutively proliferating population via a transit cell population. We questioned whether it is possible to explain our findings without inferring the existence of a second population of cells within the subependyma (i.e., relativelyquiescent stem cells).A single population model predicts that the multipotent spheres isolated in vitro are derived from theconstitutively proliferating population within the subependyma. One way to test this hypothesis is to label the constitutively proliferating cells in vivo with a replication-deficient retrovirus encoding IacZ, then look for the presence of IacZpositive spheres in vitro. Initial experiments to test this prediction reveal that the most restrictive feature of this experiment is the low infection rate of the subependymal cells with the retrovirus. Animals that received daily injections of retrovirus into the lateral ventricles (1 ~1 per injection) for 5-7 days revealed a maximum of 5% labeling of the proliferating subependymal cells. Approximately 600 spheres were isolated from over 40 such brains, and none of the spheres were IacZ positive (unpublished data). If constitutively proliferating cells are the source of spheres formed invitro,wewould haveexpected upto30spheres(5%) to be retrovirally labeled. The fact that no retrovirally labeled spheres were observed supports the conclusion that the constitutively proliferating population is not the primary source of multipotent spheres in vitro. Indeed, if the long-term labeling of less than 1% of the subependymal cells with BrdU after 31 days is an indication of the labeling of stem cells, then the likelihood of infecting a sphere-forming stem cell in vivo would be <0.05% (1% of the 5% retrovirally labeled cells). We would then expect 0.3 of 600 spheres in vitro to be retrovirally labeled. Such extrapolations indicate that the negative findings (no retrovirally labeled spheres in vitro) are difficult to interpret, although they are consistent with the proposed two population model. The most direct evidence against

Neuron 1080

the one population model, i.e., that the constitutively proliferating cells are the source of stem cells in vitro, isthefindingthatdepletingtheconstitutivelyproliferating cells in vivo does not result in a loss of stem cells isolated in vitro. As well, the relatively short survival of individual constitutively proliferating cells and the long-term depletion of the constitutively proliferating subependymal cells after a double kill (days 0 and 2) support the two population model (Figure 7). The two population model predicts that retrovirally labeled spheres would be produced by injections of retrovirus into the lateral ventricle in animals that received a single kill with 3H-thy. After a single kill, the stem cells are mitoticallyactive and should be infected with the retrovirus. Subsequent dissection and culturing of the cells should result in retrovirally labeled spheres in vitro. Once again, these experiments are limited by the low infection rate of the retrovirus in vivo; however, experiments are currently underway to test this prediction. The capacity to give rise to differentiated progeny is one of the defining criteria for a stem cell (Hall and Watt, 1989; Potten and Loeffler, 1990). The primitive hemopoietic stem cell gives rise to cells of the myelomonocytic, megakaryocytic, and erythroid lineages in the blood. The neural stem cell from the adult forebrain subependyma gives rise to glial and neuronal lineages when isolated in vitro (Reynolds and Weiss, 1992). Recently, Lois and Alvarez-Buylla (1993) have demonstrated that the constitutively proliferating subependymal cells in vivo can differentiate into neurons and glia in explant cultures. It is not known whether individual constitutively proliferating cells are lineage restricted (i.e., neurons or glia) or bipotential precursors (neurons and glia). Another definingfeatureof stem cells is their capacity for long-term self-renewal (Hall and Watt, 1989; Potten and Loeffler, 1990). Studies in vitro reveal that stem cell-generated spheres have the capacity to generate new spheres and that this procedure can be repeated indefinitely (Reynolds and Weiss, 1992; and unpublished data). Since the relatively quiescent subependymal cells give rise to spheres in vitro, we infer that these relatively quiescent stem cells which generate spheres have the capacity for self-renewal. Self-renewal in vivo is difficult to test directly. However, the size of the subependyma is maintained throughout the adult life of the animal, and because clones of constitutively proliferating cells die out over time, there must be continual long-term self-renewal within the population that replaces the constitutively proliferating cells. After a 3H-thy double kill on days 0 and 2, the proliferating subependymal population did not repopulate tocontrol values, even after 31 days in vivo.This result suggests that, once stem cells have been lost, it is impossible to regenerate the full in vivo complement of constitutively proliferating cells. It is also possible that 31 days is not a sufficient time to repopulate completely after such a severe loss of stem cells. Alterna-

tively, the failure to observe complete repopulation may reflect a positional restriction on the stem cells. For example, if the stem cells are stationary within the subependyma, then the stem cells remaining after the 3H-thy double kill may not be able to repopulate relatively distant regions within the subependyma, either because they do not receive proliferation signals over longer distances or because their progeny cannot migrate the longer distances necessaryto repopulate the adult forebrain subependyma completely. EGF (Reynolds and Weiss, 1992) has been shown to be essential for proliferation of neural stem cells in vitro. The presence of EGF-R on both constitutively proliferating as well as some relatively quiescent subependymal cells in vivo suggests that EGF may play an important role in the in vivo regulation of the proliferation of both of these populations, yet an in vivo source of ECF in the adult brain has not been clearly established. Transforming growth factor a (TGFa, a structural homologueof EGF) has its functional effects by acting on the EGF-R (Massague, 1983; Marquardt et al., 1984). TCFa mimics the effects of EGF on neural stem cells in vitro (Reynolds and Weiss, 1992), and there is immunological and molecular evidence supporting TGFa synthesis in both the developing and mature brain (Kudlow et al., 1989; Brown et al., 1990; Fallon et al., 1990). This suggests that TGFa could be the functional ligand in vivo. We have demonstrated that the subependyma of the adult mammalian forebrain contains a population of relatively quiescent stem cells that have the capacity to repopulate the constitutively proliferating subependymal cells after damage. When isolated in vitro, these stem cells have the capacity to self-renew as well as to give rise to neurons and glia (Reynolds and Weiss, 1992). In vivo, these su bependymal neural stem cells may serve as endogenous sources of the precursors for new neurons and/or glia in the adult mammalian forebrain. Experimental

Procedures

lmmunohistochemistry Adult male CD1 mice (25-30 g) were obtained from Charles River. A polyclonal sheep antibody to ECF-R (UBI) and a polyclona! rabbit antibody against nestin (a gift from Dr. R. McKay; Lendahl et al., 1990) were used. Animals were sacrificed with an overdose of sodium pentobarbital and transcardially perfused with 50-75 ml of 10% formalin (anti-EGF-R antibody) or 4% paraformaldehyde in 0.1 M phosphate buffer (nestin). The brains were removed, postfixed in paraformaldehyde containing 20% sucrose, and stored at 4°C. For EGF-R staining, 30 vrn thick coronal sections were cut through the lateral ventricles, from the rostra1 forebrain to the third ventricle. Sections were collected in primary antibody (1:200) diluted in washing solution (0.1 M phosphate-buffered saline [PBS] containing 1% normal horse serum and 0.3% Triton X-100) overnight at 4’C. The following day, the sections were washed three times (IO min each) and incubated in a biotinylated donkey anti-sheep IgC (Sigma) at I:50 for 1 hr at room temperature. After three washes, sections were incubated in avidin-conjugated fluorescein isothiocynate (FITC; I:50 dilution;Cappel)forl hrat room temperature.Thiswasfollowed by three washes. Sections were mounted on gelled slides and coverslipped with immu-mount (Shandon). Nestin staining was

Neural 1081

Stem Cells in the Adult

Subependyma

done in both rats (adult male Wistar; 250-300 g; Charles River) and mice. Rats were sacrificed as above and perfused with 150 ml of4% paraformaldehyde. Coronal brain sections (15 urn thick) were cut on a cryostat and mounted directly onto gelatin-coated slides. The slides were washed three times in wash buffer (0.1 M PBS containing 1% normal goat serum and 0.3% Triton X-100) and then incubated overnight in the anti-nestin antibody (I:1000 diluted in 10 mM PBS with 0.3% Triton X-100) at 4OC. The same protocol used for the ECF-R staining was then followed, usingabiotinylatedgoatanti-rabbitsecondan/antibody(Cappel; 1:50; diluted in wash buffer) and the avidin-conjugated FITC. The slides were coverslipped with glycerol:water (10~1). Cells immunoreactive for EGF-R and nestin were examined under a fluorescence microscope with 470 nm wavelength illumination.

Brain Dissection and Isolation of Spheres In Vitro Adult mouse brains were removed under sterile conditions following cervical dislocation. Hippocampus, cortex, striatum excluding subependyma, and striatum including subependyma were dissected and dissected into 1 mm sections. The cells were dissociated enzymatically and cultured in the presence of EGF, as described previously by Reynolds and Weiss (1992). After 8 DIV, the number of spheres formed in the culture wells was counted for each of the dissected regions. Data were collected from four independent culture preparations; brain regions from two adult mice were pooled in each of the four independent cultures.

Regional Comparison of the Number of Proliferating Cells In Vivo and the Formation of Spheres In Vitro Mice were injected with BrdU (Sigma; 65 mg/kg, dissolved in 0.007 N NaOH in 0.9% NaCI) every 2 hr for 10 hr and sacrificed 0.5 hr after the last injection tn = 4). Animals were killed by an anesthetic overdose and transcardially perfused with 4% paraformaldehyde as described above. The brains (including the olfactory bulb) were removed and kept at 4OC overnight in paraformaldehyde containing 20% sucrose. Coronal sections (30 urn) through the entire brain were cut from the olfactory bulb to the fourth ventricle. Every fifth section was collected in wells with 0.1 M phosphate buffer (4OC). The sections were incubated in 1 M HCI for 30 min at 60°C to denature the DNA. This was followed by three IO min washes with washing solution (0.1 M PBS containing 1% normal horse serum and 0.3% Triton X-100). Sections were incubated for 48 hr (4OC) in a primary mouse monoclonal antibody (1:25) directed against single-stranded DNAcontaining BrdU (Becton-Dickinson). Following incubation in the primary antibody, the same protocol as described above was followed using a biotinylated horse anti-mouse secondary antibody (Dimension Lab; 1:50) and the avidin-conjugated FITC. Sections were mounted on gelatin-coated slides, coverslipped with immu-mount, and examined under a fluorescent microscope. The total number of BrdU-immunopositive cells in every third section was counted and corrected (Abercrombie, 1946), thus revealingthetotal number of proliferating cells in the brain. For regional analysis, the brain was divided into four regions: olfactory bulb, rostra1 striatum from the rostra1 tip of the brain to the optic chiasm, caudal striatum extending caudally from the optic chiasm and including tissue up to the rostra1 fourth ventricle,andfourthventricleextendingintothe rostra1 medulla. An estimate of the total number of proliferating cells in each of the above regions was calculated. The same four areas were dissected for in vitro analysis of the sphere formation. Brains were removed, and tissue including the subependymal area was dissected from each of the four regions. Tissue from each region was dissociated enzymatically and cultured in the presence of EGF (Reynolds and Weiss, 1992). The number of spheres was counted after 8 DIV in each of the wells(2-5wellsplatedperregion perbrain),and thetotalnumber of spheres per brain region was averaged over the number of animals (olfactory bulb, n = 6; rostra1 striatum, n = 10; caudal striatum, n = 8; fourth ventricle, n = 2).

3H-Thy Kill Paradigms Adult male CD1 mice received a series intraperitoneal injections of ‘H-thy (0.8 ml per injection; 1.0 mCi/ml; spec. activity 45-55 Ciimmol; ICN Biomedical) on day 0 (three injections, one every 4 hr) to kill the constitutively proliferating subependymal cells (Becker et al., 1965; Lajtha et al., 1969). On day 0.5, 1, 2, 4, 6, or 8, animals received two intraperitoneal BrdU injections (Sigma; 1 hr apart; 65 mg/kg, dissolved in 0.007 N NaOH in 0.9% NaCI) and were sacrificed 0.5 hr after the last BrdU injection. Control mice received only the BrdU injections at the time of sacrifice. Animals were sacrificed, and the tissue was processed for BrdU immunohistochemistry as described above. The number of BrdU-labeled cells was counted in the dorsolateral corner of the lateral ventricle in sections extending from the most caudal portion of the genu of the corpus callosum to the level of the crossing of the anterior commissure. The counts were averaged from six to eight sections per animal, with a minimum of four animalspertimepoint.Theaveragenumberof proliferatingcells per 350 pm2 was then expressed as a percentage of the number of BrdU-labeled cells in the control animals (100%). In a second group of animals, ‘H-thy injections were given on day 0 (three injections, one every 4 hr) followed by an identical series of injections on day2 or4. Animals that received injections on days 0 and 2 were sacrificed on day 10 or 31. Animals that received injections on days 0 and 4 survived to day 12. Prior to sacrifice, the animals were given two injections of BrdU and were sacrificed 0.5 hr later as described above. The brains were processed for BrdU, and the number of immunoreactive cells was counted as above. Formation of Spheres In Vitro following lH-Thy Kill In Vivo Mice were divided into four groups (n = 6 per group). The first group of animals received a series of “H-thy injections on day 0 (three injections, one every 4 hr; see above). The second and third groups of animals received the same series of SH-thy injections on day 0, followed by a second series of injections on day 2 or 4. The fourth group of animals received vehicle control injections of physiological saline instead of ‘H-thy. Animals from all groups were sacrficed by cervical dislocation 16-20 hr following the last series of injections. Brains were removed, and the subependyma surrounding the lateral ventricles in the forebrain was dissected and cultured as described above. Cells from each brain were plated into two 35 mm wells. After 8 DIV, the total number of spheres was counted in each well, totaled per brain, and averaged over the number of animals. Acknowledgments We thank Dr. Ron McKay for the anti-nestin antibody. This work was supported by the Medical Research Council of Canada and the Canadian Networks of Centres of Excellence. S. W. is an Alberta Heritage Foundation for Medical Research scholar and an MRC scientist. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement”in accordance with 18 USC Section 1734 solely to indicate this fact. Received

August

9, 1994; revised

August

31, 1994.

References Abercrombie, microtome

M. (1946). Estimation of nuclear sections. Anat. Rec. 94, 239-247.

population

from

Altman, J. (1963). Autoradiographic investigation of cell proliferation in brains of rats and cats. Anat. Rec. 745, 573-591. Altman, J., and Das, G. D. (1965). Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J. Comp. Neural. 724, 319-336. Bayer, S. A., Yackel, J. W., and Pun, P. S. (1982). Neurons rat dentate gyrus granular layer substantially increase juvenile and adult life. Science 276, 890-892.

in the during

Neuron 1082

Becker, A. J., McCulloch, E. A., Siminovitch, L., and Till, J. E. (1965). The effect of differing demands for blood cell production on DNA synthesis by hemopoietic colony-forming cells of mice. Blood 26, 296-312. Brown, P. I., Lam, R., Lakshmanan, J., and Fisher, D. A. (1990). Transforming growth factor alpha in developing rats.Am. J. Physiol. 259, 256-260. Cattaneo, E., and McKay, R. (1990). Proliferation and differentiation of neuronal stem cells regulated by nerve growth factor. Nature 347, 762-765.

Potten, C. S., and Loeffler, M. (1990). Stem cells: attributes,cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt. Development 770, 1001-1020. Raedler, E., and Raedler, A. (1978). Autoradiographic study of early neurogenesis in rat cerebral cortex. Anat. Embryol. 754, 267-312. Reynolds, B.A., and Weiss, S. (1992). Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255, 1707-1710.

Chadwick,M., and Rogers, W. I. (1972).Thephysiological disposition of 5-fluorouracil in mice bearing solid L 1210 lymphocyte leukemia. Cancer Res. 32, 1045-1056.

Reynolds, B. A., and Weiss, S. (1993). EGF-responsive stem cells in the mammalian central nervous system. In Restorative Neurology and Neuroscience, Volume 6, Neuronal Cell Death and Repair, A. C. Cueilo, ed. (Amsterdam: Elsevier), pp. 247-255.

Craig, C. C., Morshead, C. M., Roach, A., and van der Kooy, D. (1994). Evidence for a relatively quiescent stem cell in the adult mammalian forebrain. J. Cell Biochem. 78 (Suppl.), 176.

Smart, I. (1961). The subependymal layer of the mouse brain and its cell production as shown by radioautography after thymidineH’ injection. J. Comp. Neural. 776, 325-338.

Fallon, J. H., Annis, C. M., Gentry, L. E., Twardzik, D. R., and Laughlin, S. E. (1990). Localization of cells containing transforming growth factor-alpha precursor immunoreactivity in the basal ganglia of the adult rat brain. Growth Factors 2, 241-250.

Smart, I., and LeBlond, C. P. (1961). Evidence for division transformations of neuroglia in the mouse brain, as derived radioautography after injection of thymidine-J-t?. J. Comp. rol. 776, 349-367.

Hall, P. A., and Watt, F. M. (1989). maintenance of cellular diversity.

Spangrude, C. J., Smith, L., Uchida, N., Ikuta, K., Heimfeld, Friedman, J., and Weissman, I. L. (1991). Mouse hematopoietic stem cells. Biood 78, 1395-1402.

Stem cells: the generation and Development 706, 619-633.

Harrison, D. E., and Lerner, C. P. (1991). Most primitive hematopoietic stem cells are stimulated to cycle rapidly after treatment with 5-fluorouracil. Blood 78, 1237-1240. Kaplan, M. S., and Hinds, rat: electron microscopic ence 797, 1092-1094.

J. W. (1977). Neurogenesis in the adult analysis of light radioautography. Sci-

Kiefer, R., Lindholm, D., and Kreutzberg, kin-6 and transforming growth factor-81 rat facial nucleus following motoneuron rosci. 5, 775-781.

G. W. (1993). InterleumRNAs are induced in axotomy. Eur. J. Neu-

Korn, A. P., Henkelman, R. M., Ottensmeyer, F. P., and Till, J. E. (1973). Investigations of a stochastic model of haemopoiesis. Exp. Hemat. 7, 362-375. Kudlow, J. E., Leung, A. W. C., Kobrin, M. S., Paterson, A. J., and Asa, S. L. (1989). Transforming growth factor-a in the mammalian brain. lmmunohistochemical detection in neurons and characterization of its mRNA. j. Biol. Chem. 264, 3880-3883. Lajtha, L. A., Pozzi, L. V., Schofield, properties of haemopoietic stem

R., and Fox, M. (1969). Kinetic cells. Cell Tiss. Kinet. 2,39-49.

Lendahl, U., Zimmerman, L. EL., and McKay, R. D. C. (1990). CNS stem ceils express a new class of intermediate filament protein. Cell 60, 585-595. Lois, C., and Alvarez-Buylla, A. (1993). Proliferating subventricular zone cells in the adult mammalian forebrain can differentiate into neurons and glia. Proc. Natl. Acad. Sci. USA 90,2074-2077. Lois, C., and Alvarez-Buylla, A. (1994). Long-distance neuronal migration in theadult mammalian brain. Science264,1145-1148. Luskin, M. B. (1993). Restricted proliferation postnatally generated neurons derived from ventricular zone. Neuron 77, 173-189. Marquardt, H., Hunkapiller, G. 1. (1984). Rat transforming relation to epidermal growth

and migration of the forebrain sub-

M. W., Hood, L. E., and Todaro, growth factor type 1: structure and factor. Science 223, 1079-1082.

Massague, J. (1983). Epidermal growth factor-like growth factor. II. Interaction with epidermal growth tors in human placenta membranes and A431 cells. 258, 13614-13620. McConnell, S. K. (1988). the mammalian cerebral

Development cortex. Brain

transforming factor recepJ. Biol. Chem.

and decision-making Res. Rev. 73, l-23.

in

Morshead, C. M., and van der Kooy, D. (1992). Postmitotic death is the fate of constitutively proliferating cells in the subependymal layer of the adult mouse brain. J. Neurosci. 72, 249-256. Okano, H. J., Pfaff, D. W., and Gibbs, R. B. (1993). expression in brain: correlations with 3H-thymidine tion and neurogenesis. J. Neurosci. 73, 2930-2938.

RB and Cdc2 incorpora-

and from NeuS.,

Streit, W. J., and Kreutzberg, G. W. !1988). Response of endogenous glial cells to motor neuron degeneration induced by toxic ricin. J. Comp. Neurol. 268, 248-263. Sturrock, R. R., and Smart, I. H. M. (1980). A morphological study of the mouse subependymal layer from embryonic life to old age. J. Anat. 730, 391-415. Wen, J. Y. M., Morshead, C. Satellite cell proliferation in results from the release of a sensory neurons. J. Cell Biol.

M., and van der Kooy, D. (1994). the adult rat trigeminal ganglion mitogenic protein from explanted 724, 1005-1015.