Multipotent progenitor cells in the adult dentate gyrus

Multipotent Progenitor Cells in theAdult Dentate Gyrus

Fred H. Gage, Gerd Kempermann, Theo D. Palmer, Daniel A. Peterson, Jasodhara Ray

Laboratory of Genetics, The Salk Institute, 10010 N. Torrey Pines Rd., La Jolla, CA 92037

Accepted 31 March 1998

be genetically marked and transplanted back to theABSTRACT: Neurogenesis persists in the adultadult brain, where they survive and differentiate intodentate gyrus of rodents throughout the life of themature neurons and glial cells. Although multipotentorganism. The factors regulating proliferation, sur-stem cells exist in the adult rodent dentate gyrus, theirvival, migration, and differentiation of neuronal pro-biological significance remains elusive. q 1998 Johngenitors are now being elucidated. Cells from the adultWiley & Sons, Inc. J Neurobiol 36: 249–266, 1998.hippocampus can be propagated, cloned in vitro, andKeywords: neurogenesis; FGF-2; hippocampus; steminduced to differentiate into neurons and glial cells.cells; transplantationCells cultured from the adult rodent hippocampus can

Most neurons of the adult central nervous system ence the rate of proliferation, the extent of survival,are terminally differentiated, exist through the life the substrates of migration, and the events control-of the organism, and, when they die, are not re- ling fate choice decisions (Reynolds and Weiss,placed. However, evidence exists that small popula- 1990, 1992; Reynolds et al., 1992; Vescovi et al.,tions of neurons continue to be born in the adult 1993; Gage et al., 1995a; Kilpatrick et al., 1995,subventricular zone and hippocampus. In particular, 1996; Temple et al., 1995, 1996; Ray et al., 1997a;adult hippocampal granule cells continue to be born McKay, 1997).throughout the life of the rodent, and the process As studies of regulation and generality of adultof neurogenesis, which includes proliferation, sur- neurogenesis continue, a recurring question remainsvival, migration, and differentiation, can be regu- as to the functional significance of these newly bornlated by a variety of stimuli. Although Altman first neurons. Paralleling the in vivo characterization ofobserved the proliferative potential of the adult ro- adult neurogenesis, in vitro studies have revealeddent brain in 1962 (Altman, 1962), the significance that the adult brain contains cells that can be inducedof progenitor cells and the generality of their occur- to proliferate in culture and retain some capacityrence have been controversial (Rakic, 1985). The for differentiation. Basic fibroblast growth factorrecent development of methods for cell imaging, (FGF-2) can induce hippocampal progenitor cellslabeling, and counting has made possible the accu-

to proliferate in vitro, and these proliferating cellsrate and quantitative assessment of in vivo adult

can be cloned and induced to differentiate into glialneurogenesis. In addition, several laboratories have

cells and neurons in vitro. The exact molecular path-begun to reveal some of the factors that can influ-

ways leading to the fate choice decisions are onlynow beginning to be understood. Whether the path-ways are exactly the same as those followed duringCorrespondence to: F. H. Gage

Contract grant sponsor: NINDS embryonic development, or whether adult neuro-Contract grant sponsor: NIA genesis is somehow unique remains to be deter-Contract grant sponsor: APA

mined.Contract grant sponsor: NFPq 1998 John Wiley & Sons, Inc. CCC 0022-3034/98/020249-18 Several studies have now shown that cells of

249

8p45 1988/ 8p45$$1988 06-29-98 17:50:26 nbioa W: Neurobio

250 Gage et al.

the adult and fetal nervous system as well as fetal- genitor cells’’ is used to describe this populationbelow.derived cell lines can be isolated and characterized,

expanded indefinitely in vitro, genetically labeledin vitro, and transplanted back into the adult central Proliferation and Differentiation in thenervous system, where they can survive and differ- Adult Rodent Dentate Gyrusentiate in a target-specific manner into neurons(Gage et al., 1995a; Brustle and McKay, 1996; Experiments using the proliferation marker bromo-

deoxyuridine (BrdU) to label dividing cells in theFisher, 1997; Martınez-Serrano and Bjorklund,1997; Stemple and Mahanthappa, 1997). The first subgranular zone show that hundreds of cells within

this thin lamina divide daily. In the days followingpart of this review summarizes the results obtainedfrom studies that track the activity and fate of the BrdU treatment, the number of labeled cells de-

creases by two thirds over the next 4 weeks (Kemp-endogenous proliferating hippocampal progenitorscells. Next, we summarize current findings on the erman et al., 1997a) via either cell death or contin-

ued proliferation that renders the BrdU undetect-ability of FGF-2 to act as a mitogen for rat hippo-campal progenitor cells and the conditions that in- able. About half of the cells in which BrdU is still

detectable develop a neuronal phenotype, migratefluence the survival and fate of these cells. Finally,we end with a review of transplantation studies at- into the granule cell layer, and become morphologi-

cally indistinguishable from the other surroundingtempting to determine how similar adult-derivedhippocampal cells isolated in vitro are to the endog- granule cells [Figs. 1(B) and 3(A)]. About 15%

of the cells differentiate into glial cells [Fig. 3(B)].enous adult granule cells of the dentate gyrus. Thissummary provides an update on our present knowl- The remaining 35% of cells do not show a detect-

able neuronal or glial phenotype up to 4 weeks afteredge of the existence, behavior, and potential ofadult hippocampal progenitors, while suggesting ad- cell division (the time point commonly investigated

in our experiments) [Fig. 3(C)] . This latter groupditional questions that will require further investiga-tion. of cells supposedly consists of (a) differentiated

cells of a phenotype that was not investigated, (b)cells that are postmitotic and well advanced in theprocess of differentiating but do not yet express

NEUROGENESIS IN THE ADULT specific marker proteins of mature cells, (c) quies-HIPPOCAMPUS cent cells at any stage of development that might

ultimately divide again or differentiate, (d) lineage-restricted progenitor cells, and/or (e) stem cells.There are two areas of the adult brain that continue

to generate neurons throughout adult life: the sub- Although exact kinetic studies are lacking todate, a conservative estimate from our own studiesventricular zone of the anterior lateral ventricles and

the subgranular zone in the dentate gyrus of the in mice shows that the rate of ongoing adult hippo-campal neurogenesis is not negligible: a 3-month-hippocampus. In the adult dentate gyrus, cells divide

continuously in a thin lamina between the hilar re- old mouse produces, per day, at least 1 neuron/2000 existing granule cells (Kempermann et al.,gion and the granule cell layer of the dentate gyrus,

i.e., the subgranular zone [Fig. 1(A)] . In analogy 1997a). The rate of neurogenesis decreases withincreasing age, but even in old animals the sameto stem cell concepts from hematology and embry-

ology, and based on the in vitro findings described process takes place (Kuhn et al., 1996). In accor-dance with earlier reports on rats (Bayer et al.,later in this review, a theory has emerged that char-

acterizes this population, as follows. Stem cells of 1982), our results demonstrate that the total granulecell number increases into midlife (6 months) ofthe central nervous system may divide asymmetri-

cally. Some of the proliferative cells are probably mice and then reaches a plateau, but does not de-crease (Kempermann et al., 1998). This findingself-renewing stem cells that sustain the pool of

proliferative cells (Fig. 2) . Asymmetric divisions suggests that hippocampal neurogenesis continu-ously builds up the granule cell layer into midlifeproduce one daughter cell that enters the pathway

leading to differentiation. These lineage-restricted and then maintains an equilibrium between produc-tion of new cells and neuronal loss. The extent andcells may continue to divide, and to date, it is not

known to what degree the detectable population of rate of cell death in the dentate gyrus have not yetbeen satisfactorily determined. Cell death might oc-dividing cells in the subgranular zone consists of

true stem cells or lineage-restricted progenitor cells. cur in two different cellular populations: (a) amongthe undifferentiated progenitor cells that do notBased on this uncertainty, the cautious term ‘‘pro-


Adult Neurogenesis 251

Figure 1 Proliferation (A) and survival (B) of progenitor cells in the subgranular zone of3-month-old mice. The inserts show that cells labeled with the proliferation marker bromodeox-yuridine (BrdU), which integrates into the DNA, have an irregular shape and dense andhomogeneous chromatin at 1 day after the last injection of BrdU and are more rounded, witha chromatin structure that resembles that of mature granule cells at 4 weeks after BrdU injection.Note the decrease in the number of BrdU-positive cells between 1 day and 4 weeks afterinjection. Scale bar: 75 mm (25 mm for the inserts) . (From Kempermann et al., Proc. Natl.Acad. Sci. USA 94:10409–10414, copyright (1997) National Academy of Sciences, U.S.A.,reproduced with permission).

progress down a lineage, and (b) among the existing rate of proliferation greatly decreases with increas-differentiated granule cells. Taken together, these ing age, whereas the percent survival of newbornresults imply that adult hippocampal neurogenesis cells remains constant. In addition, the ratio of cellsis a process that requires an intricate and complex differentiating into neurons decreases significantlyregulation (Fig. 2) . Neurogenesis is not an all-or- with age (Kempermann et al., 1998). The molecularnothing event that can be sufficiently described by mechanisms underlying these different regulatorythe division of the neuronal progenitor cell alone. events remain to be determined.The new neuron stands at the end of a series of Hormones, neurotransmitters, and growth factorsdistinct steps, all of which apparently underlie sepa- are among the mediators that appear to be involvedrate regulation. We use the term ‘‘neurogenesis’’ in in the regulation of adult hippocampal neurogenesis.this broader sense, wherein birth of a new neuron Although a variety of hormones might influenceis at the end rather than at the beginning of the adult hippocampal neurogenesis, only the effects ofsequence of events defining neurogenesis. Among glucocorticoids have been examined in detail. Thethe steps that can be readily identified are prolifera- results from several studies show that glucocorti-tion, survival, migration, differentiation, and estab- coids have an inhibitory effect on neurogenesislishment of functional connections. Comparing dif- (Gould et al., 1992; Gould, 1994). Proliferation in-ferent inbred mouse strains, we found that the creases in adrenalectomized rats, and this effect canstrains differed from each other based not only on

be antagonized by systemic application of glucocor-these different regulatory steps, but also in the net

ticoids (Gould et al., 1992). The effects on otherproduction of new neurons (Kempermann et al.,

levels of the regulation of neurogenesis remain to1997a). This result suggested that several genesbe investigated.contribute to the regulation of adult hippocampal

Manipulation of the glutamatergic input to theneurogenesis.granule cell layer also influences adult hippocampal

Cellular and Molecular Events Affecting neurogenesis (Cameron et al., 1995; McEwen,Neurogenesis 1996). Glutamatergic deafferentiation caused an in-

crease in neurogenesis, with a marked effect on pro-Some factors affecting known regulatory levels ofneurogenesis have been identified. For example, the liferation, although survival, migration, and differ-


252 Gage et al.

entiation have not yet been examined. Furthermore, hippocampal neurogenesis have also been investi-gated. Recombinant growth factors have beenit has been reported that treatment with the gluta-

mate receptor antagonist MK-801 increases prolif- chronically infused into the ventricular system ofadult rats (Craig et al., 1996; Kuhn et al., 1997).eration in the subgranular zone (Gould et al., 1994).

In apparent contrast to these results is the finding Epidermal growth factor (EGF), which had a strongeffect on proliferation in the subventricular zone,that experimental temporal lobe seizures induced by

excitatory amino acids cause a dramatic increase in did not cause a significant increase in labeled divid-ing cells in the subgranular zone (Kuhn et al.,proliferation (Parent et al., 1997; Bengzon et al.,

1997). The newborn neurons formed under these 1997). Surprisingly, EGF did not cause more ofthese cells to survive, but induced a significant phe-conditions, however, elaborated aberrant connec-

tions. Normally, the new granule cells extend neu- notypic shift leading to more astrocytes and fewerneurons. FGF-2, which stimulated proliferation, sur-rites along the mossy fiber pathway to CA3, provid-

ing the potential to establish new functional connec- vival, and neuronal differentiation in the subventri-cular zone/olfactory bulb, had none of these effectstions (Stanfield et al., 1988; Parent et al., 1997). A

recent study reported that neurogenesis in the tree in the hippocampus, although FGF-2 might not havediffused well enough intraparenchymally to reachshrew is regulated by an interaction between psy-

chosocial stress and N-methyl-D-aspartate (NMDA) the progenitor cells in the subgranular zone fromthe ventricular infusion site (Kuhn et al., 1997).receptor activation (Gould et al., 1997), suggesting

a complex interaction between normal and excit- However, after systemic delivery, FGF-2 has beenshown to increase cell proliferation in neonatal ratatory effects on adult neurogenesis.

The effects of various growth factors on adult hippocampus and cerebellum through an as yet un-known mechanism (Tao et al., 1996).

Very little is known to date about the moleculesthat are important for migration of newborn cellsin the dentate gyrus. However, the region-specificexpression of polysialylated neural cell adhesionmolecules (PSA-NCAM), whose expression is in-

Figure 2 The different steps that constitute neurogen-esis. Cells proliferate in the subgranular layer and theirsurviving progeny migrate into the granule cell layerproper, where neuronal differentiation is completed. Theopen arrows indicate points at which regulation mightoccur. It is not known whether the proliferating cellslabeled by bromodeoxyuridine incorporation are truestem cells or already lineage-determined precursor cells.The existence of both cell types in the subgranular zoneremains hypothetical. In contrast to their in vitro counter-part, neuronal progenitor cells in vivo to date are solelycharacterized retrospectively as cells that proliferate dur-ing adulthood and have progeny that acquire neuronalcharacteristics after division. As the number of labeledproliferating cells decreases with increasing time afterlabeling, it is assumed that newborn cells are eliminatedby cell death. As apoptotic elimination has not yet beendirectly demonstrated, these steps are marked with a ques-tion mark in the drawing. Although the newborn cellshave been shown to send out neurites along the mossyfiber tract (Stanfield and Trice, 1988; Parent et al., 1997),functional integration of the new neurons remains as yetundemonstrated. It is therefore marked with a questionmark. Multipotent stem cells will also give rise to glialcells, which is not depicted in this drawing.



volved in the migration of neuroblasts in the devel- vivo and the regulatory events acting upon the neu-ral stem and progenitor cells in neurogenic regionsoping brain, has been shown in both the dentate

gyrus and the olfactory rostral migratory stream of of the adult brain is incomplete. Once a functionfor hippocampal neurogenesis is determined, it willthe adult central nervous system (CNS) (Bonfanti

et al., 1992; Seki and Arai, 1993; Kuhn et al., 1996). be possible to establish whether the activity of neu-ronal stem cells in the adult CNS is a curious excep-Newly generated neurons from almost the entire

subventricular zone (SVZ) proceed to the olfactory tion to the rule of no new neurons in adult brainsor an integral part of normal adult brain function.bulb through the rostral migratory stream and even-

tually integrate into the granule and glomerular celllayers (Luskin, 1993; Doetsch and Alvarez-Buylla,1996). Work by Alvarez-Buylla and coworkers PROLIFERATION AND

DIFFERENTIATION OF ADULT(Rousselot et al., 1994; Lois et al., 1996) has char-acterized a process called chain migration with ex- PROGENITOR CELLS IN VITROpression of PSA-NCAM as a molecule that may aidthe migration. The generation of new neurons in the The discovery of the presence of neuronal progeni-

tor cells in the adult brain allowed us to predict andadult dentate gyrus also correlates with the expres-sion of PSA-NCAM on their surface and their mi- subsequently demonstrate that these proliferating

populations of cells can be isolated and induced togration into the granule cell layer (Seki and Arai,1993; Kuhn et al., 1996). These observations sug- divide and differentiate into neurons and glial cells

in vitro. The studies of adult hippocampal progeni-gest that one may reasonably expect the regulatorycues present in the adult CNS to mirror those ex- tor cells in vitro were based on our initial observa-

tions that FGF-2 was a mitogen for fetal hippocam-isting in the developing CNS, albeit probably witha more restricted expression. The presence of PSA- pal tissue (Ray et al., 1993). In the following sec-

tion, we review the effects of FGF-2 on fetalNCAM in areas of neurogenesis in the adult brainsupports its importance there. PSA-NCAM expres- hippocampal cells, which leads into the use of very

similar conditions to culture adult cells and charac-sion decreases with increasing age (Bonfanti et al.,1992), but the interaction with other regulatory terize them.molecules has not yet been investigated. In addition,most studies of PSA-NCAM have focused on the FGF-2–Responsive Progenitor Cells inolfactory system (see this volume of the Journal of the Fetal Rat BrainNeurobiology) .

The identification of cytokines and growth factorsthat have survival and mitogenic effects on stem orThe Functional Significance ofprogenitor cells has been pivotal in their isolationNeurogenesis in Adult Hippocampusand culture (Reynolds and Weiss, 1990, 1992;Reynolds et al., 1992; Vescovi et al., 1993; GageDespite the growing evidence of a complex regula-

tory apparatus for adult hippocampal neurogenesis, et al., 1995a; Kilpatrick et al., 1995; Temple et al.,1995, 1996; Gritti et al., 1996; Ray et al., 1997a).surprisingly little is known about the function of the

newborn neurons. We have been able to demon- One of the peptide growth factors that has bothtrophic and neurite elongation effects on brain cellsstrate that challenging adult mice by housing them

under the stimulating conditions of an enriched en- is FGF-2 (Walicke et al., 1986). In situ mappingstudies have shown that large amounts of FGF-2vironment results in a greater number of new neu-

rons (Kempermann et al., 1997b) (Fig. 4) . This are expressed in all regions of the developing brain,suggesting an important role for this growth factorincrease in neurons was not due to any detectable

effect on proliferation of progenitor cells, but the in early neurogenesis (Liu and Nicoll, 1988; Gonza-les et al., 1990; Gomez-Pinilla et al., 1994). Lownumber of surviving newborn cells increased sig-

nificantly. In middle aged (6 months) and senescent levels of FGF-2 mRNA are detected in neurons andastrocytes throughout the adult brain; the highestmice (18 months) , an additional effect on the distri-

bution of phenotypes among the surviving newborn amounts are present in the neurons of the CA-2region of the hippocampus (Emoto et al., 1989;cells could be detected, which can be interpreted as

a neuron-specific selectivity of the survival-promot- Woodward et al., 1992). FGF-2 is expressed byboth cultured neurons and astrocytes (Pettmann eting effect (Kempermann et al., 1998).

In summary, the body of knowledge detailing al., 1985; Perreud et al., 1986; Ferrara et al., 1988;Hatten et al., 1988). In culture, FGF-2 has beenthe regulation of adult hippocampal neurogenesis in


254 Gage et al.



found to increase survival of neurons isolated from cord (E14) precursor cells showed that at up to aconcentration of 1 ng/mL, FGF-2 has survival effects,hippocampus and other regions of embryonic rodent

brains (Morrison et al., 1986; Walicke et al., 1986; whereas at a concentration of 10 ng/mL, it functionsas a mitogen. The maximum effect was observed at aWalicke, 1988; Walicke and Baird, 1988). How-

ever, the trophic and neurite elongation effects of concentration of 20 ng/mL, irrespective of the regionsfrom which the cells were isolated (Ray et al., 1993;FGF-2 are concentration dependent. The minimal

concentration needed for neuronal survival is be- Ray and Gage, 1994). Concentrations higher than 20ng/mL showed no increase in proliferative effect. Us-tween 10 and 30 pg/mL; the concentration needed

for process outgrowth ranges between 200 and 500 ing FGF-2 at its maximum effective concentration,long-term progenitor cell cultures were establishedpg/mL (Walicke et al., 1986). Subsequent studies

have shown that at a higher concentration (5 ng/ from embryonic rat hippocampus, spinal cord andbasal forebrain (Ray et al., 1993; Ray and Gage, 1994;mL), FGF-2 exhibited short-term mitogenic effects

on neuronal precursor cells isolated from different Minger et al., 1996). These cells were maintainedthrough multiple passages for months and can be fro-regions of mouse and rat CNS (Genesberger et al.,

1987; Murphy et al., 1990; Deloulme et al., 1991; zen down for long-term storage and recultured as nec-essary (Ray et al., 1993; Ray and Gage, 1994). Pri-Drago et al., 1991). Thus, FGF-2 can exert a variety

of effects on the same or different cell populations mary, passaged, and recultured cells grew attached tothe substratum (polyornithine and laminin) and notin culture, but a specific function of the growth

factor is determined by its concentration. as floating balls (neurospheres). Examination of anumber of neurotrophins and growth factors withExamination of the dose-dependent effects of FGF-

2 on embryonic rat hippocampal (E16) and spinal known survival and mitogenic properties (Drago et

Figure 3 (A–C) Phenotypes of the surviving newborn (BrdU-positive) cells 4 weeks afterlabeling with BrdU. The confocal microscopic images of sections stained with triple immuno-fluorescence show (A) two BrdU-positive cells (yellow) that are also positive for the granulecell marker calbindin (red), (B) a BrdU-positive cell in the subgranular zone that is colabeledwith antibodies against the astrocytic marker glial fibrillary acidic protein (GFAP) (blue) , and(C) a BrdU-positive cell that shows colocalization for neither calbindin nor GFAP. The latterpopulation supposedly contains the surviving stem and progenitor cells. (From Kempermannet al., Proc. Natl. Acad. Sci. USA 94:10409–10414, copyright (1997) National Academy ofSciences, U.S.A., reproduced with permission.) (D–F) Adult hippocampal progenitor cellsexpressed a variety of early markers in culture. (D) Although the immature markers A2B5(green) and nestin (red) were primarily expressed in separate cell populations, some cellsexpressed both markers (yellow). (E) More mature glial markers such as O4 (green) andGFAP (red) were observed in nonoverlapping cell populations. (F) Neuronal markers such asMAP-5 (green) were localized to a subset of NSE-positive cells (red) . Cultures in (D–F)were counterstained with DAPI (blue) . (G, H) Cultured adult hippocampal progenitor cells 8weeks after grafting to the adult hippocampus. (G) Adult hippocampal progenitor cells labeledwith the BrdU prior to engraftment survived well within the dentate gyrus. (GCLv-granule celllayer ventral) . (H) Four weeks following engraftment, adult hippocampal progenitor cellsmarked in culture with the LacZ transgene demonstrated integration into the granule cell layer(GCL) of the dentate gyrus and showed complex dendritic arborization that is characteristicof endogenous granule neurons following immunocytochemical detection of b-Gal. [(D–H)From Gage et al., Proc. Natl. Acad. Sci. USA 92:11879–11883, copyright (1997) NationalAcademy of Sciences, U.S.A., reproduced with permission.] (I–K) Adult hippocampal progeni-tor cells at 8 weeks after being grafted into the adult olfactory bulb. Adult hippocampalprogenitor cells marked with BrdU (green) were able to migrate into the olfactory bulb glomeru-lar cell layer, where they expressed calbindin (red) [(I) arrow] or tyrosine hydroxylase (blue) ,[(J) arrow; BrdU colocalization appears as light blue against the tyrosine hydroxylase] . Theexpression of tyrosine hydroxylase does not occur in adult hippocampal neurons, suggestingthat the grafted adult hippocampal progenitors responded to local cues leading to the expressionof this phenotype. (K) Grafted BrdU-labeled (green) adult hippocampal progenitor cells alsocontributed to the olfactory granule cell layer, where they expressed NeuN (red) but not GFAP(blue) . [(I–K) Reprinted by permission from Nature, Suhonen et al., Nature 383:624–627,copyright 1996 Macmillan Magazine Ltd.]


256 Gage et al.

Figure 4 Enriched environment (A) in which 12 mice lived for 40 or 68 days, and a standardcage (B) for three control animals at the same scale [scale bar in (A) for (A) and (B) Å25 cm]. At 4 weeks after discontinuation of BrdU injection, enriched living mice (D) hadsippgnificantly more surviving newborn (BrdU-positive) cells than controls (C); quantificationis in (E) (details are in Kempermann et al., 1997a). Scale bar in (D) for (C) and (D) Å 80mm. (Reprinted with permission from Nature, Kempermann et al., 386:493–495, copyright1997 Macmillan Magazine Ltd.)

al., 1991; Reynolds et al., 1992; Svendsen et al., 1995) and calretinin (neurons present in the hippocam-pus) , glia fibrillary acidic protein (GFAP), and ga-showed that at a concentration of 20 ng/mL, nerve

growth factor (NGF), brain-derived neurotrophic fac- lactocerebroside (Gal C). Spinal cord and basalforebrain cultures contained a few motoneuronstor (BDNF), and neurotrophin-3 (NT-3) had no pro-

liferative effects on embryonic spinal cord progenitor (Ray and Gage, 1994) and cholinergic neurons(Minger et al., 1996), respectively. These resultscells, whereas epidermal growth factor (EGF) exhib-

ited mitogenicity, albeit at a much lower level than may indicate that most cells in culture are multipo-tent progenitors not yet committed to a specific lin-that observed with FGF-2 (Ray and Gage, 1994).

However, a different concentration or a combination eage. In addition, the presence of phenotype-specificneurons in distinct brain region cultures may indi-of factors may be more effective.

Immunocytochemical analysis of the nature of cate inherent differences between FGF-responsiveprogenitor cells present in different regions of thecells present in passaged cultures from fetal rat hip-

pocampus, spinal cord, or basal forebrain showed brain. However, these cells are found in small num-bers.the presence of progenitor, neuronal, and glial cells

(Ray et al., 1993; Ray and Gage, 1994; Minger A notable difference between primary progeni-tors and their proliferative counterparts grownet al., 1996; Ray et al., 1997a). Many cells were

immunoreactive for the neural progenitor marker through multiple passages was observed in studiesof voltage and ligand-gated currents in fetal rat hip-Nestin, an intermediate filament protein associated

with germinal centers in the developing brain (Len- pocampal progenitors and the regulation of thesecurrents by chronic exposure to FGF-2 or otherdhal et al., 1990). A majority of cells also coex-

pressed neuronal markers (neuron-specific enolase, growth factors (Sah et al., 1997a). Growth of pri-mary cells under proliferative conditions (in FGF-microtubule-associated protein-5, and neurofila-

ments) . Only a few cells progressed toward a ma- 2) was associated with low-sodium, calcium,NMDA, and kianate currents. However, neuronalture neuron, astrocyte, or oligodendrocyte, as

judged by the number of cells expressing calbindin markers were strongly up-regulated in equivalent



cultures by factors such as BDNF, NT-3, serum, Schachner, 1981, 1982). The more mature neuronalmarkers such as NeuN were not expressed by theseKCl, or growth on glial monolayers. After multiple

passages, sodium, calcium, and NMDA currents de- cells, whereas a small number (ú5%) expressedcalbindin and calretinin, proteins normally ex-clined further. Growth of cells in BDNF and NT-

3 had little effect on sodium and calcium current pressed by hippocampal neurons. Only a small pop-ulation (õ2%) of cells differentiated into matureexpression. Interestingly, kianate and g-aminobu-

tyric acid (GABA) responses remained moderate astrocytes and oligodendrocytes, as determined bythe expression of GFAP and Gal C. Thus, most cellsin multipassaged cultures and were unaffected by

the environmental conditions used. These results in- in culture are uncommitted and coexpress markersusually attributed to specific neuronal and glial lin-dicate that proliferative neural progenitors grown in

FGF-2 respond to environmental conditions to a eages, which may be a unique property of the pro-genitor cells.much lesser extent than their primary counterparts.

This decrease in responsiveness may be due to the The isolation of progenitor cells from adult hip-pocampus and the fact that neurogenesis is restrictedundifferentiated state of most of the cells present in

the passaged cultures and may suggest that more in the discrete germinal zones within the adult CNShave raised the question of whether the lack of neu-complex differentiation conditions than those exam-

ined in this report may be needed to restore all the rogenesis in most regions is due to the absence ofprogenitor cells or to the loss of specific neurogenicfunctional currents in these cells.signals. To address the first part of the question,attempts were made to isolate, culture, and charac-FGF-2–Responsive Progenitor Cells interize progenitor cells from other adult brain regionsthe Adult Rat Brain(Palmer et al., 1995) using the same conditions em-ployed in adult hippocampal cultures (Gage et al.,Although as most regions of the mammalian brain

mature there is a decrease in cell proliferation, mi- 1995b). Progenitor cells were cultured from neuro-genic regions—the hippocampus and subventricu-gration, and differentiation as well as a lack of re-

generative responses to injury, neurogenesis persists lar zone—and non-neurogenic regions—the sep-tum and striatum. Neurogenic regions yielded morein the granule cell layer of the hippocampus, sub-

ventricular zone, and olfactory system even in adult- proliferative cells and higher numbers of coloniescontaining progenitor-like cells (39% for hippocam-hood, as described above. The presence of FGF-2–

responsive neural progenitor cells in the adult rat pus and 40% for subventricular zone) compared tonon-neurogenic regions (20% for septum and stria-hippocampus was examined by culturing these cells

(Gage et al., 1995b) using the same approach em- tum). In addition, cells isolated from non-neuro-genic regions initially grew more slowly than thoseployed to isolate, propagate, and characterize pro-

genitor cells from fetal brain (Ray et al., 1993). from proliferative zones. This difference disap-peared after two to three passages and progenitorDuring the first 2 months after plating, cells attached

to the substratum and a mixed population of cells cell cultures with similar growth rates (doublingtime 36–48 h) were established from all areas. Cul-emerged that divided slowly. Repeated passaging

gave rise to a more restricted population containing tures from all regions contained virtually indistin-guishable populations of progenitor, neuronal, andcells with round to oval somata and thin processes.

Like the fetal brain-derived progenitor cells, these glial cells. As was seen in a previous study withhippocampal cells (Gage et al., 1995b), most cellscells can be maintained in culture through multiple

passages, and freeze-thawed and recultured. in subventricular zone, septum, and striatum cul-tures expressed progenitor cell markers, coex-Immunocytochemical analysis showed that a va-

riety of progenitor, neuronal, and glial markers were pressed lineage specific markers and gave rise tofew differentiated neurons or glia (Palmer et al.,expressed by these cells in culture, and, interest-

ingly, most cells expressed markers representing all 1995).of the lineages [Fig. 3(D–F)]. A majority of cellsexpressed Nestin and MAP-2c, a microtubule-asso- FGF-2–Responsive Neural Progenitorsciated protein found in both immature neurons and in the Adult Rat Spinal Cordglia (Tucker, 1990; Przyborski and Cambray-Dea-kin, 1995). One unusual characteristic of FGF-2– The isolation of FGF-2–responsive progenitor cells

from quiescent regions of the brain has raised theresponsive progenitors is the expression of O4, aganglioside epitope first identified in cells des- question of whether similar cell populations can be

found in the adult rat spinal cord and, more specifi-tined to become oligodendrocytes (Sommer and


258 Gage et al.

cally, whether they reside at all levels of the cord. Clonal Multipotent Progenitor Cells fromAdult Rat Hippocampus GenerateThe existence of neural progenitor cells in cervical,Neurons and Gliathoracic, lumbar, and sacral areas of adult rat spinal

cord was shown by culturing these cells (Shihabud-Cultures of neural progenitors derived from thedin et al., 1997), employing the same approach andadult hippocampus initially contain a variety of cellmethodology used to culture progenitors from adulttypes at different stages of growth. Some proliferatehippocampus (Gage et al., 1995b). Although allrapidly in response to FGF-2. Other, more differen-areas generated rapidly proliferating progenitors,tiated cells proliferate slowly or not at all. Withinthe cultures were heterogeneous in nature and thethe first few passages, the slower-growing cells arecell morphologies varied within a given region aslost in favor of the rapidly dividing progenitors, andwell as among regions. Like progenitors culturedthe populations are almost exclusively made up offrom brain regions (Gage et al., 1995b; Palmer etundifferentiated cells.al., 1995), the major population in all cultures con-

Many of the cells within the progenitor culturessisted of small, phase-bright, round cells with multi-can be induced to differentiate through the simpleple small to medium thin processes and a minorremoval of FGF-2 and/or following contact-depen-population of large phase-bright as well as largedent growth arrest (Palmer et al., 1997). Just thephase-dark cells. The frequency of different cellact of slowing culture growth may allow some ofmorphologies varied between regions. The cervicalthe spontaneously differentiating cells to acquireregion contained only a few flat cells, whereas theyrecognizable markers; however, by altering the cul-were more abundant in lumbar and sacral areas. Inture conditions, the accumulation of cells in glial oraddition, cervical and thoracic cultures grew fasterneuronal lineages can be dramatically influenced.than lumbar and sacral cultures. Immunohistochem-For example, density-dependent growth arrest in theical analysis showed that while the majority of cellspresence of reduced FGF-2 (5 ng/mL vs. 20 ng/

cultured from cervical, thoracic, and lumbar regionsmL), which is used to maintain proliferative pro-

expressed the progenitor marker vimentin, onlygenitors, produces populations that are predomi-Ç20% cells in sacral culture were vimentin positive.nantly oligodendrocytes. Growth arrest in the ab-

Quantitative determination of the percentage of cells sence of FGF-2 yields few oligodendrocytes andexpressing specific lineage markers also showed dif- instead produces both neurons and astrocytes. Fi-ferential expression among regions. nally, replacing FGF-2 with fetal bovine serum

Spinal cultures were further characterized for (FBS) produces populations that are mostly astro-their ability to generate motoneurons. About 2–8% cytes.of cells in different regional cultures were motoneu- The ability to generate all three neural lineagesrons, as judged by their large cell bodies (ú20 m) from these bulk cultures raises two possibilities. Ei-and expression of low-affinity NGF receptor (p75). ther separate glial and neuronal progenitors wereThese results suggest that progenitor cells exist in present in the cultures and/or a single populationdifferent levels of the adult spinal cord, and a small of multipotent progenitors produced all three neuralnumber of them can spontaneously differentiate into lineages. To investigate these possibilities, a retrovi-neurons and glial cells under culture conditions. In ral marking strategy was used to mark individualaddition, progenitors from all regions of the spinal cells, and clonal populations derived from thesecord are able to give rise to motoneurons. Thus, cells were expanded and evaluated for the types ofalthough progenitor cells are present in different cells they were able to generate. While proliferatingareas of the CNS (Gage et al., 1995b; Palmer et al., at log phase, the clonal populations contained few1995; Shihabuddin et al., 1997) and their overall differentiated cells and expressed the same imma-morphology and characteristics are the same, as ture markers as seen in the nonclonal cultures. Whenjudged by their expression of lineage-specific mark- induced to differentiate, the clonal populations dis-ers and their potentiality to differentiate into neu- played the same tendency to generate mostly oligo-rons and glia, there may be inherent differences dendrocytes, mixtures of neurons and astrocytes, orbetween them. For example, spinal cord progenitors mostly astrocytes, depending on the culture condi-can produce motoneurons that cannot be generated tions. In addition, each of these differentiated popu-by forebrain progenitors. These results are similar to lations carried the same retroviral integration site asthose seen with fetal CNS-derived progenitor cells the initial rapidly dividing clone, thus demonstratingwhich, although similar in many respects, can give that all three lineages were generated from the same

progenitor. Several of the clones were subsequentlyrise to region-specific neurons.



marked with a second retroviral vector and sub- Two classes of bipotent cells were isolated fromfetal human progenitor cells immortalized with thecloned. These subclones also retained the ability to

generate all three lineages, suggesting that many of tet-regulatable vector system (Sah et al., 1997b).In the absence of tet, proliferating cells expressedthe progenitors from the adult hippocampus had the

stem cell–like properties of proliferative self re- the progenitor cell marker. In the presence of tet,one cell line, B4, differentiated only into neurons,newal and the potential to generate progenitors for

multiple neural lineages. as judged by its expression of neuronal markers.However, horse or rat serum or phorbol ester (PMA,an activator of protein kinase C) treatment overrodeAdult Brain-Derived Progenitorthe cells’ normal default fate and differentiated themPopulation Contains Cells Committed tointo astrocytes. Another group of clones exhibitedSpecific Lineagesboth neuronal and astrocytic default differentiationpathways in the presence of tet. Again, serum treat-Although the study by Palmer et al. (1997) reported

that clonal progenitors were multipotent, no lineage- ment in the presence of tet generated more astro-cytes and suppressed neuronal differentiation. Onrestricted progenitor populations were identified in

this study. Thus, it is not clear whether the pluripo- the other hand, forskolin (FSK), which activatesadenyl cyclase, induced more of these cells to differ-tent cells directly generate all types of neural cells or

whether separate populations of lineage-restricted entiate into neurons. However, FSK and horse se-rum had more pronounced effects on this group ofcells give rise to specific cell types. This issue can

be addressed by oncogenic immortalization, which cells than on B4 cells. In contrast, PMA was mosteffective in promoting astrocytic differentiation inarrests cells at specific stages of development and

prevents their terminal differentiation (Cepko, B4 cells. These clonal cell lines could not be differ-entiated into oligodendrocytes under any of the con-1988; Lendhal and McKay, 1990). To isolate cells

arrested at different stages of development which ditions tested. These results suggest that bulk cul-tures of progenitor cells not only contain pluripotentcan be further differentiated without apoptotic cell

death, a retroviral vector expressing v-myc onco- progenitor cells but also cells at different develop-mental stages. Although their intrinsic signals dif-gene in a regulatable fashion was used (Hoshimaru

et al., 1996; Sah et al., 1997b). In this system, a ferentiate them into a specific lineage(s) , the extrin-sic signals can override these cues and alter theirtetracycline (tet)-controlled transactivator strongly

activates transcription from human cytomegalovirus ultimate fate.(CMV) minimal promoter fused to a tet operatorsequence in the absence of the antibiotic. However,even low concentrations of tet (0.01–1.0 mg/mL) Proliferative Effects of Other Memberscan completely abolish the transcription activation of the FGF Family on Neural Progenitorby the transactivator. Adult rat hippocampus or fetal Cellshuman brain-derived progenitor cells were infectedwith this recombinant retrovirus and clonal popula- Fibroblast growth factor-2 is a member of an FGF

family composed to date of 13 related but geneti-tions were isolated and characterized (Hoshimaruet al., 1996; Sah et al., 1997b). In the absence of cally distinct polypeptides. Although there is 30–

55% sequence homology between the members,tet, clonal rat hippocampus-derived cell line HC2S2remained in a proliferative state, and cells were their functions vary widely. Examination of some

of the family members (FGF-1, FGF-2, and FGF-polygonal in shape with very small processes (Ho-shimaru et al., 1996). All cells in culture expressed 4 to FGF-7) for their mitogenic effects on neural

progenitor cells from embryonic and adult rat hippo-v-myc and the progenitor cell markers, but not theneuronal or astrocytic marker proteins. Suppression campus showed that although FGF-1 had a small

effect, FGF-4 was as effective as FGF-2 (Ray etof myc expression by tet treatment was sufficientto make proliferating cells exit the cell cycles and al., 1997b) [Fig. 5(A)] . In addition, FGF-4 showed

similar dose-dependent mitogenic effects on neuralundergo terminal differentiation. Differentiatedcells became phase-bright, extended processes and progenitor cells as FGF-2. At a concentration of

¢10 ng/mL, FGF-4 was a proliferative factor, withexpressed mature neuronal markers. The differentia-tion of HC2S2 cells into mature neurons was further 20 ng/mL being the optimum concentration. Other

FGFs tested were nonmitogenic. The mitogenic ef-confirmed by their expression of large sodium andcalcium currents and firing of regenerative action fects of FGF-2 could not be increased further by

combining with FGF-4 [Fig. 5(B)] , indicating thatpotentials.


260 Gage et al.

Figure 5 Effects of FGF family members on [3H]thymidine incorporation by embryonichippocampal progenitors. (A) Although FGF-1 showed some incorporation, FGF-2 and FGF-4 had the highest effects. The values for FGF-5 to FGF-7 were similar to that of control (noFGF). (B) Combinations of FGF-2 and FGF-4 (20 ng/mL) or FGF-2 and FGF-5 did notincrease or decrease the thymidine incorporation levels of FGF-2 alone, indicating that FGF-2 and FGF-4 may be acting through the same receptor(s) . (C) Sequence of FGFs in the 10-amino acid region. Amino acids not common between FGF-2 and FGF-4 are boxed. The aminoacid sequence of second receptor-binding domain of FGF-2 is represented as FGF 2-2. (D)Effects of peptides on thymidine incorporation by adult hippocampal progenitors. Both peptidesP2 (FGF-2) and P4 (FGF-4) inhibited the mitogenic effects of FGF-2, but peptides P1 (FGF-1) or P5 (FGF-5) had no effect (Ps / FGF-2). Addition of FGF-2 prior to the addition ofpeptides P2 and P4 (FGF-2 / P2 or P4) abolished their inhibitory effects. (From Ray et al.,Proc. Natl. Acad. Sci. USA 94:7047–7052, copyright 1997 National Academy of Sciences,U.S.A., reproduced with permission.)

the two growth factors may be functioning through ferent from other nonmitogenic family members.The high-affinity receptor-binding sites are founda common receptor(s) .in the two regions of the FGF-2 molecule. A com-parison of the primary sequences of FGFs showedSequence of FGF-2 Involved in Itsa remarkable similarity (9 of 10) between a 10–Mitogenic Effect on Neural Progenitoramino acid (aa) sequence of FGF-2 (aa 68–77)Cellsand FGF-4 (aa 122–131) located within the firstputative receptor-binding domain of FGF-2 (Ray etSince FGF-2 and FGF-4 function through a com-

mon receptor(s) , their primary sequences may con- al., 1997b) [Fig. 5(C)] . Synthetic peptides corre-sponding to these sequences (peptides P2 and P4,tain a common receptor-binding region that is dif-



respectively) had no proliferative effects by them- the fetal tissue prior to transplantation to enrich forneural progenitor cells. As the primary neural cul-selves on the neural progenitor cells. However, they

could block the mitogenic effects of FGF-2 [Fig. tures from fetal CNS are not clonal, but may containa variety of neural progenitors, the approach has5(D)] . In addition, chemical crosslinking studies

using 125I-FGF-2 showed specific reduction of the been refined to derive immortalized clonal cell lines,usually through introduction of an oncogene. Thebinding of the radiolabeled growth factor to its re-

ceptor(s) in the presence of P2 and P4. The speci- goal of this approach is the establishment of clonalpopulations of progenitor cells induced to remainficity of this 10-aa sequence was further supported

by the fact that the peptides containing the aa se- in continuous cell cycle without transformation. Inaddition, these cultures are amenable to infectionquence of the second putative receptor binding re-

gion of FGF-2 (aa 115–129; P2–2) or those de- with vectors containing a transgene whose productcan be expressed following engraftment of the pro-rived from other nonmitogenic FGFs (P1 and P5)

did not block the cellular proliferation by FGF-2 genitor cells.and did not prevent binding of 125I-FGF-2 to neuralprogenitor cells. Proliferation was not inhibited Transplantation of Immortalized Cells towhen cells were treated with FGF-2 before or con- the Braincurrently with the peptides [Fig. 5(D)] , indicatingthat the peptides are primarily functioning by occu- While progenitor cell lines grafted to the developing

CNS demonstrated lineage differentiation that ap-pying the high-affinity FGF-binding sites on the re-ceptor(s) present on the cells but that the peptide peared to be determined by the site of their en-

graftment (Renfranz et al., 1991; Snyder et al.,by itself has no capacity to activate the signalingpathways leading to mitogenesis. Thus, the binding 1992), placement of these cell lines in the adult

CNS results in primarily glial differentiation (Martı-of FGF-2 to its high-affinity receptor through this10-aa sequence is necessary for exerting its mito- nez-Serrano et al., 1995; Lundberg et al., 1997),

especially following brain damage (Lundberg andgenic activity.Bjorklund, 1996; Snyder and Macklis, 1996). How-ever, not all cell lines respond equivalently, asshown by the predominantly neuronal differentia-TRANSPLANTATION OF ISOLATED

ADULT PROGENITOR CELLS TO THE tion of cells from the RN33B clonal cell line follow-ing engraftment to the hippocampus (ShihabuddinADULT BRAINet al., 1995). This observation underscores the dif-ficulties inherent in the use of cell lines in regardThe ability to isolate and characterize in vitro a

population of neural progenitor cells from fetal to interpretation of the mechanisms regulating phe-notypic differentiation and their potential for thera-(Ray et al., 1993) or adult CNS (Gage et al., 1995b)

suggests that these cells may be used to repopulate peutic use. Since cell lines are established from dif-ferent embryonic tissues at different ages and subse-damaged areas of the adult brain. To realize this

possibility, the cultured progenitor cells must retain quently maintained in different culture conditionsand immortalized by different manipulations, it be-the potential to differentiate into neurons. Further-

more, these progenitor cells would need to respond comes difficult to generalize the results from variousreports in regard to the contribution of intrinsic lin-to local cues to enable them to integrate into the

appropriate regions. Finally, evidence of phenotypic eage restriction and the response to local cues inthe final phenotypic differentiation of the engraftedspecificity, synaptic connectivity, and/or functional

integration into neuronal ensembles would be neces- cells.sary to validate a potential therapeutic application.

A number of different approaches have been used Grafting Adult Progenitors to theto address the questions of lineage determination Dentate Gyrusand responsiveness to local cues by neural progeni-tor cells. These approaches have been the subject of Based upon the observation that neurogenesis per-

sists in some regions of the adult CNS, an alterna-several recent reviews (Brustle and McKay, 1996;Fisher, 1997; Martınez-Serrano and Bjorklund, tive approach has been to derive cultures of neural

progenitors from defined anatomical regions of the1997; Stemple and Mahanthappa, 1997). Unlikeearlier fetal grafting studies, where solid or dissoci- adult CNS (Gage et al., 1995b) [Fig. 3(G,H)] . Us-

ing the enriched cultures of adult hippocampal pro-ated embryonic CNS was transplanted into the adultCNS, current approaches have attempted to culture genitors, we reintroduced these cells to the intact


262 Gage et al.

adult hippocampus (Gage et al., 1995b). By mark- not expressed by neurons within the adult hippo-campus from which the AHPs were derived. Thising the progenitor cells with the thymidine analog

BrdU or with the LacZ transgene prior to grafting, study (Suhonen et al., 1996) suggests that regionalcues in the adult CNS not only can direct progeni-we were able to evaluate the distribution and differ-

entiated phenotype of marked cells within the hip- tors into a neuronal or glial lineage, but can alsodirect differentiating neurons down specific pheno-pocampus. Upon examination, approximately half

of the grafted adult hippocampal progenitors typic pathways.These data suggest that some regions of the adult(AHPs) within the CA fields failed to differentiate;

the remaining half had a glial phenotype. This result CNS (e.g., hippocampus and olfactory system) per-sist in the expression of localization and differentia-was also true for AHPs located in regions of the

dentate gyrus that had been damaged by the grafting tion cues for neuronal precursors, while many re-gions of the adult CNS (e.g., cerebellum) may notprocedure. However, of the AHPs located in the

undamaged regions of the dentate gyrus, over half continue to express these cues. In addition to theirphenotypic integration, AHPs can be geneticallyexpressed a neuronal phenotype, with the remainder

showing no discernible differentiated phenotype. modified to express a transgene (Gage et al., 1995b;Suhonen et al., 1996). The ability to insert trans-None of the AHPs in the intact dentate gyrus exhib-

ited a glial phenotype. genes into AHPs could augment their usefulness forex vivo gene delivery strategies in a way that hasalready been demonstrated for immortalized pro-Target-Directed Differentiation ofgenitor cell lines (Martinez-Serrano et al., 1995).Grafted CellsFurthermore, the ex vivo insertion of transgenes mayallow grafted AHPs to endogenously express theThese results argued for a site-specific determina-

tion of lineage differentiation in the adult CNS. necessary cues for neuronal differentiation and inte-gration in brain regions where these cues are notHowever, since these AHPs were reintroduced to

the same anatomical region from which they had otherwise expressed. However, a simpler approachmay be to cograft AHPs with other somatic cellsbeen derived, it was not clear if their neuronal dif-

ferentiation was due to internal determinants that (e.g., fibroblasts or astrocytes) that have been modi-fied to express the needed cues. Of course, suchproceeded in a permissive, anatomically appropriate

environment or whether the environment itself pro- experiments presuppose the identification of whichcues are necessary for neuronal differentiation andvided cues that were the determinants of phenotype.

To address this question, we performed a second functional integration. Further work is required toidentify the necessary cues.experiment in which labeled AHPs were grafted

either to the adult hippocampus—the homotypic The response of the adult CNS to damage mayinclude the expression of factors that may cue neuralsite—or to two heterotypic sites (Suhonen et al.,

1996): the cerebellum, which is non-neurogenic in progenitors to differentiate and integrate as part ofthe brain’s own repair mechanism. Selective focalthe adult, and the SVZ-olfactory bulb, where neuro-

genesis continues in the adult [Fig. 3(I-K)] . cortical injury has been shown to contribute to themigration and differentiation of transplanted neuralResults of the examination of labeled AHPs

grafted to the hippocampus were consistent with our precursors into pyramidal-like neurons at the site ofinjury (Macklis, 1993; Sheen and Macklis, 1995).previous results. AHPs grafted to the cerebellum

survived as well as the hippocampal grafts, indicat- The ability to manipulate these cues may permit notonly the integration of grafted neuronal precursorsing that the graft site does not influence survival.

However, the cerebellar grafted AHPs did not mi- but also the recruitment of endogenous neuronalprecursors to replace damaged neurons.grate away from the graft site and most did not

express any differentiated phenotype apart from asmall percentage that expressed a glial phenotype.In contrast, AHPs grafted into the olfactory system CONCLUSIONmigrated along the rostral migratory stream into theolfactory bulb and then radially into the granular Unexpectedly, the nervous system of adult rodents

retains immature cells that can divide, migrate, andand glomerular layers in a similar fashion to endog-enous SVZ-derived progenitors. A portion of these differentiate into neurons, astroglia, and oligoden-

droglia. The rate of proliferation and extent of dif-AHPs not only exhibited a neuronal phenotype butalso expressed tyrosine hydroxylase. Although this ferentiation depends on the location in the brain

where the cells reside, but the general phenomenonphenotype is appropriate to the olfactory bulb, it is



BRUSTLE, O. and MCKAY, R. D. (1996). Neuronal pro-appears to be a reliable part of the normal adultgenitors as tools for cell replacement in the nervousbrain biology. Considerable evidence exists now forsystem. Curr. Opin. Neurobiol. 6:688–695.the ability to induce the proliferation of cells from

CAMERON, H. A., MCEWEN, B. S., and GOULD, E. (1995).the adult nervous system. The existence of theseRegulation of adult neurogenesis by excitatory inputcells in mammalian species beyond rodents is con-and NMDA receptor activation in the dentate gyrus. J.tributing to the generalization of the neural stem/Neurosci. 15:4687–4692.

progenitor cell as a normal component of the adult CEPKO, C. L. (1988). Immortalization of neural cellsbrain. Factors such as neurotransmitters and hor- via retrovirus-mediated oncogene transduction. Trendsmones, as well as environmental stimuli, have been Neurosci. 11:6–8.shown to influence the rate of proliferation or sur- CRAIG, C. G., TROPEPE, V., MORSHEAD, C. M., REYN-

vival of these cells, but the specific cellular and OLDS, B. A., WEISS, S., and VAN DER KOOY, D. (1996).molecular events controlling adult neurogenesis re- In vivo growth factor expansion of endogenous subep-

endymal neural precursor cell populations in the adultmain to be elucidated. More importantly, even themouse brain. J. Neurosci. 16:2649–2658.most fundamental reasons why these cells exist in

DELOULME, J. C., BAUDIER, J., and SENSENBRENNER, M.the adult brain are unknown. Neurogenesis may be(1991). Establishment of pure neuronal cultures froman integral part of normal plasticity; yet, modelsfetal rat spinal cord and proliferation of the neuronalthat would accommodate their function conflict withprecursor cells in the presence of fibroblast growthexisting ‘‘hardwire’’ models of the brain and arefactor. J. Neurosci. Res. 29:499–509.

at odds with how adult neuroplasticity is presentlyDRAGO, J., MURPHY, M., CARROLL, S. M., HARVEY, R.

conceived. The adult brain appears to be more plas- P., and BARTLETT, P. F. (1991). Fibroblast growthtic than previously appreciated, and the role of adult factor-mediated proliferation of central nervous systemneurogenesis in this augmented plasticity needs to precursors depends on endogenous production of insu-be understood to incorporate the concept of cell lin-like growth factor I. Proc. Natl. Acad. Sci. USAgenesis into our current modeling of normal and 88:2199–2203.diseased brain function. The ability to culture these EMOTO, N., GONZALEZ, A.-M., WALICKE, P. A., WADA,

E., SIMMONS, D. M., SHAMASAK, S., and BAIRD, A.cells in vitro and transplant them back to the adult(1989). Basic fibroblast growth factor (FGF) in theand developing nervous system provides an im-central nervous system: identification of specific loci ofportant tool to determine the role that progenitorbasic FGF expression in the rat brain. Growth Factorscells play in the adult brain.2:21–29.

FERRARA, N., OUSLEY, F., and GOSPODAROWICZ, D.This work was supported in part by grants and con- (1988). Bovine brain astrocytes express basic fibro-

tracts from NINDS and NIA to FHG, as well as support blast growth factor, a neurotropic and angiogenic mito-from APA and NFP. The authors thank A. Alvarez-Bu- gen. Brain Res. 462:223–232.ylla, S. Temple, and M. L. Gage for their helpful com-

FISHER, L. J. (1997). Neural precursor cells: applicationsments on this article.

for the study and repair of the central nervous system.Neurobiol. Dis. 4:1–22.

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Multipotent progenitor cells in the adult dentate gyrus

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