Experimental Neurology 171, 11–21 (2001)doi:10.1006/exnr.2001.7729, available online at http://www.idealibrary.com on

Embryonic-Derived Glial-Restricted Precursor Cells (GRP Cells) CanDifferentiate into Astrocytes and Oligodendrocytes in Vivo

Juan Herrera,*,† Hui Yang,‡ Su-Chun Zhang,§ Chris Proschel,¶ Patrick Tresco,* Ian D. Duncan,§Marla Luskin,‡ and Margot Mayer-Proschel*,¶,1

*Keck Center for Tissue Engineering and †Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah; ‡Department of CellBiology, Emory University, Atlanta, Georgia 30322; §Department of Medical Sciences, School of Veterinary Medicine, University

Wisconsin, Madison, Wisconsin 53706; and ¶Center for Cancer Biology, University of Rochester, Rochester, New York

Received February 7, 2001; accepted May 8, 2001

4tgotsutv

mmadaa

We have isolated and characterized a unique glial-restricted precursor cell (GRP) from the embryonicspinal cord. Clonal analysis demonstrated that thesecells are able to generate oligodendrocytes and twodistinct type of astrocytes (type 1 and type 2) whenexposed to appropriate signals in vitro. We now showthat many aspects of these cells are retained in vivo.GRP cells are restricted to the glial lineage in vivo asthey seem to be unable to generate neuronal pheno-types in an in vivo neurogenic environment. GRP cellssurvive and migrate in the neonatal and adult brain.Transplanted GRP cells differentiate into myelin-forming oligodendrocytes in a myelin-deficient back-ground and also generate immature oligodendrocytesin the normal neonatal brain. In addition, GRP cellsalso consistently generated glial fibrillary protein-ex-pressing cells in the neonatal and adult brain, a prop-erty not consistently expressed by other glial precur-sor cells like the O-2A/OPC cells. We suggest that thelineage restriction of GRP cells and their ability togenerate both oligodendrocytes and astrocytes invivo together with their embryonic character thatallows for extensive in vitro expansion of the popula-tion makes the cell useful for clinical application.© 2001 Academic Press

Key Words: glial precursor; spinal cord; embryonic;transplantation; lineage restriction; oligodendrocytes;astrocytes.

INTRODUCTION

We have recently identified a novel glial-restrictedprecursor cell (GRP cell) that can be isolated from theembryonic rat spinal cord (32, 33). A number of char-

1 To whom correspondence should be addressed at Center for Can-cer Biology, KMRB, University of Rochester MC, 601 Elmwood Av-enue, Box 633, Rochester, NY 14642. Fax: (716) 273-1450. E-mail:margot_mayer-proschel@urmc.rochester.edu.

11

acteristics distinguish GRP cells from other glial pre-cursor cells. Most importantly, GRP cells can differen-tiate into type 1 astrocytes, type 2 astrocytes, andoligodendrocytes. In contrast, oligodendrocyte–type 2astrocyte (O-2A) progenitor cells only generate type 2astrocytes and oligodendrocytes, while previously iden-tified astrocyte precursor cells do not generate oligo-dendrocytes (26, 37). GRP cells also further differ fromany other glia precursor cells in their lack of expressionof platelet-derived growth factor (PDGF) receptor aand their dependence on basic fibroblast growth factor(bFGF) as a survival factor (33). In addition, GRP cellscan be expanded for a prolonged time in tissue culturewithout losing their differentiation potential. The char-acterization of GRP cells is thus far based on single cellin vitro experiments. While the in vitro system in itselfcan be highly defined and can produce reliable andreproducible data, it may not faithfully represent thein vivo situation. This is a particular concern for theanalysis of glial precursor cells due to the generalfailure of primary O-2A progenitor cells to generateastrocytes following transplantation in vivo. Interest-ingly, however, cell lines generated from O-2A cellsseem to be able to generate astrocytes in vivo (1, 7, 14,1). As the transplantation of transformed or immor-alized cells raises concerns about the possibility of theraft to initiate or participate in tumor formation, it isf particular interest to evaluate primary cells in aransplantation paradigm. As the study of GRP cellshould ultimately yield insight into the possibility ofsing cell transplantation as a tool of clinical interven-ion, the behavior of these cells in the more complex inivo environment needs to be evaluated.We conducted a series of transplantation experi-ents aimed at characterizing the differentiation andigration of GRP cells in vivo. We show that many

spects of the differentiation potential of GRP cellsescribed in vitro are retained in vivo in the neonatalnd adult rat brain. Transplantation of GRP cells inton appropriate neurogenic region did not yield cells of

0014-4886/01 $35.00Copyright © 2001 by Academic Press

All rights of reproduction in any form reserved.

twdama

iosr

12 HERRERA ET AL.

a neuronal phenotype, confirming the lineage restric-tion seen in vitro. To determine whether GRP cellsgenerate myelin-producing cells, we utilized an animalmodel (md rats) for the hereditary human myelin dis-order Pelizaeus–Merzbacher disease (PMD) (18, 45).Md rats carry a mutation in the X-linked gene encodingproteolipid protein (PLP) and do therefore not generateCNS myelin. The absence of endogenous myelin allowsthe conclusion that PLP-positive myelin seen post-transplantation is generated by the grafted cells. Thisallows for rapid screening of graft-derived myelin-pro-ducing cells without the need to specifically label graftcells. Using this model, we show conclusively that GRPcells can differentiate into myelin-producing cells invivo. In addition, transplantation of GRP cells intovarious regions of the neonatal and adult rat brainsuggests that GRP cells can generate glial fibrillaryacidic protein (GFAP)-expressing cells.

MATERIAL AND METHODS

Isolation and purification of GRP cells. Stablegreen fluorescent protein (GFP) expressing GRP cellswere generated in vitro using recombinant plnx-retro-virus encoding GFP. Retrovirus was produced and ap-plied to target cells as previously described by Pear andcolleagues (30). Briefly, Phoenix cells were transfectedwith the retroviral construct using a calcium phos-phate transfection procedure. Viral supernatant col-lected from the Phoenix cells was applied to purified

1

ed by GFP–GRP Cells Transplanted into the SVZa4 Days Posttransplantation

Grafted GFP–GRP cells labeled by

Anti-GFAP Anti-PLP

RMS OB SVZp CC CTX RMS OB SVZp CC CTX

1 1 1 1 1 2 2 2 2 21 1 1 1 1 2 2 2 2 21 1 1 1 1 2 2 2 2 21 1 1 1 1 2 2 2 2 21 1 1 1 1 2 2 2 2 21 1 1 1 1 2 2 2 2 21 1 1 1 1 1 1 1 1 11 1 1 1 1 1 1 1 1 11 1 1 1 1 1 1 1 1 11 1 1 1 1 1 1 1 1 11 1 1 1 1 1 1 1 1 11 1 1 1 1 1 1 1 1 11 1 1 1 1 1 1 1 1 11 1 1 1 1 1 1 1 1 1

rmined by double labeling sections in which anti-GFP was used toonal specific type b-III-tubulin), or anti-GFAP to identify astrocytes, rostral migratory stream; OB, olfactory bulb; SVZp, posteriorbrillary acidic protein; GFP, green fluorescent protein; GRP, glial

FIG. 1. The differentiation potential of GRP cells is not impaired bythe expression of green fluorescent protein (GFP). Purified GRP cellswere infected with a GFP-expressing retrovirus and cultured for 2 days.Successfully transfected cells were harvested by FACS using GFP as asorting marker. The homogenous GFP-expressing cells were cultured indefined basal medium supplemented with either 10 ng/ml PDGF 1hyroid hormone (T3) or 20 ng/ml BMP-2. Cells exposed to PDGF 1 T3ere stained after 7 days with anti-GalC (A and B) to visualize oligo-endrocytes. Cells exposed to BMP-2 were stained after 5 days withnti-GFAP (C and D). GFP-expressing cells differentiated in the sameanner as uninfected cells in that they generated oligodendrocytes and

strocytes in the predictable manner.

TABLE

A Summary of the Cell-Type-Specific Markers Expressof 14 Neonatal Rats 3, 7, and 1

Animalcase No.

Age atimplantation

Age atperfusion

TuJ1

RMS OB SVZp CC CTX

GRP1 P1 P4 2 2 2 2 2GRP2 P1 P4 2 2 2 2 2GRP3 P1 P4 2 2 2 2 2GRP10 P1 P4 2 2 2 2 2GRP11 P1 P4 2 2 2 2 2GRP13 P1 P4 2 2 2 2 2GRP4 P1 P8 2 2 2 2 2GRP5 P1 P8 2 2 2 2 2GRP6 P1 P8 2 2 2 2 2GRP12 P1 P8 2 2 2 2 2GRP14 P1 P8 2 2 2 2 2GRP15 P1 P8 2 2 2 2 2GRP16 P1 P15 2 2 2 2 2GRP17 P1 P15 2 2 2 2 2

Note. The phenotypes of the transplanted GFP–GRP cells were detedentify the GRP cells in conjunction with either TuJ1, recognizing neurr PLP (proteolipid protein) to identify early oligodendrocytes. RMSubventricular zone; CC, corpus callosum; CTX, cortex; GFAP, glial fiestricted progenitors.

Paf

13IN VIVO CHARACTERIZATION OF GLIAL-RESTRICTED PRECURSORS

GRP cultures. Successfully transfected A2B51/GFP1

cells (using a double labeling procedure) were sorted byfluorescent activated cell sorter (FACS). Clearly dou-ble-positive A2B5/GFP cells were grown in DMEM/F12-BS supplemented with 10 ng/ml bFGF and 10ng/ml PDGF-AA until a cell population of 60,000 cells/transplant was obtained. Prior to and shortly aftertransplantation an aliquot of the cells was stained withanti-GFAP. We only evaluated cases in which GRP cellpopulations were transplanted that did not contain anyGFAP1 cells prior and shortly after the transplanta-tion procedure in order to ensure that the graft culturehas not begun to differentiate.

Immunocytochemistry for cell cultures. Staining forthe cell surface markers A2B5 (9) (which labels GRPcells and other glial progenitor cells) and anti-galacto-cerebroside GalC (15) (which labels oligodendrocytes)was carried out in cultures of living cells or cells fixedfor 10 min with 4% paraformaldehyde (PFA). For stain-ing using antibodies against intracellular antigens likeGFAP (2) cultures were fixed with ice-cold methanol.Secondary antibodies were coupled to either fluores-cein or rhodamine.

Transplantation of GFP-labeled GRP cells into theneonatal subventricular zone (SVZ). Prior to trans-

FIG. 2. GRP cells express glial phenotypes following transplantathe neonatal SVZa was analyzed using antibodies against neuron

LP-DM20 (C–E) to identify oligodendrocytes. For consistency, anti-GFP and a rhodamine conjugated secondary to recognize each orom the occipital cortex visualized with a dual FITC/rhodamine fi

b-tubulin. (B) A representative fluorescent photomicrograph from tFITC/rhodamine filter, shows numerous GFAP-positive host cells (rGFAP (yellow, arrows), as well as GFP (1)/GFAP (2) cells (green)callosum stained with anti-GFP (C) and anti-PLP-DM20 (D). The imalso express this oligodendrocyte marker (arrows). (CC, corpus callozone.) Bars, 100 mm.

plantation, purified GFP–GRP cells were centrifugedand resuspended in DMEM/F-12 with 10% fetal calfserum (FCS), and their viability (usually greater than95%) was determined using trypan blue. The final con-centration of the GFP–GRP cells used for transplanta-tion was 1 3 104 cells/ml. Using the coordinates de-scribed previously (51), 3 ml of the cell suspension wasinjected into the right anterior portion of the neonatalrat forebrain anterior subventricular zone (SVZa) of 14rat pups at postnatal day P1. The neurogenic SVZarepresents a region distinct from the remainder of theSVZ and shows a characteristic lack of astrocyte-spe-cific gene expression in the postnatal animals, al-though the neuron-specific isoforms of tubulin (a1 andb-III tubulin) are expressed abundantly in the SVZaand RMS (21). The animals were anesthetized by hy-pothermia for 5 min and positioned in a Sylgard mold.The pups were covered under ice throughout the pro-cedure to keep them anesthetized. Using a dissectingmicroscope, an incision was made in the skin along themidline of the skull, and a small piece of bone over theright forebrain was elevated and reflected. The piamater was removed from the underlying developingcerebral cortex and a Hamilton syringe loaded with 3ml GFP-labeled GRP cells (1 3 104 cells/ml) was in-

into the SVZa. The phenotype of GFP–GRP cells transplanted intocific type b-III -tubulin (A), GFAP (B) to identify astrocytes, andrescein-conjugated secondary antibody was used to visualize thee other markers. (A) A representative fluorescent photomicrographdemonstrates that the GRP cells (arrows) do not express type IIIsubventricular zone posterior to the SVZa, visualized with a dual, interspersed with transplanted GRP cells, many of which express–E) Representative fluorescent photomicrographs from the corpuses are superimposed in E, showing a subset of GFP–GRP cells that; CTX, cortex; LV, lateral ventricle; SVZp, posterior subventricular

tion-spefluof th

lterheed). (Cag

sum

sakaarmm

imatpob

iwb(Psw(datsct

14 HERRERA ET AL.

jected. Animals were returned to their mothers afterbeing revived under a heat lamp.

Transplantation of GFP-expressing GRP cells intothe adult SVZa and corpus callosum (CC). Male Fi-cher 344 rats (250–280g, 10–11weeks old) were useds hosts. The rats were anesthetized with a mixture ofetamine (65 mg/kg), xylazine (7.5 mg/kg), andcepromazine (0.5 mg/kg) at a dose of 0.1 ml/100 gnimal weight. The animal scalp was fixed into a ste-eotaxic apparatus (Lab Standard stereotaxic instru-ent, Steolting) with the plane of the incisor bar 23.2m below the interaural line. A 500-mm burr hole was

drilled at 1.5 mm anterior and 3.0 mm lateral tobregma specific for SVZ transplants. The GFP–GRPcells were drawn into a 30-gauge needle using a 1-mlHamilton syringe and introduced into the hole at adepth of 5 mm from the surface of the brain. Threemicroliters of the suspension (roughly 60,000 cells) wasinjected over 3 min and the needle was in place for 5min to prevent backflow from cells. CC transplantswere performed at 1.5 mm anterior and 3.5 mm lateralto bregma and 4.0 mm deep from the surface of thebrain. The scalp was closed using a 3.0-mm suture.Control rats underwent sham transplantation proce-dures delivering vehicle (phosphate-buffered saline,PBS) only. To avoid the use of immunosuppression, weused the inbred rat strain Fischer 344 for the trans-plantation into the adult. A total of 10 rats received acell graft into the SVZa from which 5 animals wereanalyzed after 7 days and 5 animals after 28 days.Likewise, a total of 10 animals received craft cells intocorpus callosum of which 5 were analyzed after 7 daysand 5 after 28 days.

To determine the GRPs ability to generate myelin-producing oligodendrocytes, purified GRP cells weretransplanted into dorsal column of the spinal cord of8-day-old md rats as described (48). Specifically, ninemd rats and two control rat received about 50,000 cellsin 1 ml. Two weeks following transplantation animalswere sacrificed and analyzed.

Tissue processing and immunofluorescence of trans-plants. Neonatal transplants were immunostained at3, 7, or 14 days after injection of GRP cells. The exper-imental animals were anesthetized by ether andtranscardially perfused with freshly made 4% PFA in0.1 PBS (pH 7.4). The brains were removed, postfixedin 4% PFA, cryoprotected in PBS with 20% sucrosesolution (in PBS), embedded in OCT compound, andstored at 280°C until sectioned. Ten-micrometer sag-ttal sections of the brain were cut on a cryostat and

ounted on Superfrost Plus slides (Fisher) and storedt 220°C immunostained. To determine the final posi-ion of the transplanted cells, single-label ICC waserformed using anti-GFP on sections from the brainsf the experimental animals. Labeled cells could alsoe detected without anti-GFP labeling but the signal

ntensity was lower. After thawing the sections, theyere rinsed in 0.1 M PBS (pH 7.4) for 30 min andlocked with PBS containing 10% normal goat serumPBS–NGS). Anti-GFP (Quantum) was diluted 1:500 inBS–NGS containing 0.4% Triton X-100, placed on theections, and incubated overnight at 4°C. The slidesere rinsed in 0.1 M PBS. A fluorescein isothiocyanate

FITC)-conjugated goat anti-mouse secondary antibodyiluted in PBS–NGS was then placed on the sectionsnd kept at room temperature (RT) for 2 h. The sec-ions were rinsed in PBS and covered using Vecta-hield. Double labeling with anti-GFP and one ofell-type-specific markers listed below was performedo evaluate the phenotype of the transplanted GFP1

GRP cells. The markers used were anti-GFAP (Dako,diluted 1:500) to identify astrocytes, anti-PLP-DM20(Chemicon, diluted 1:200) to identify oligodendrocytesand TuJ1 (BabCO, diluted 1:2000) that recognize typeIII b-tubulin to identify neurons. FITC-conjugated sec-ondary antibody was used to enhance the signal of the

FIG. 3. Distribution of GRP cells after transplantation into theneonatal SVZa. Shown is the pattern of distribution of GFP-labeled GRP cells in the forebrain 3 days after transplantationinto the SVZa. Cells were identified by their expression of GFP.The dotted lines represent the injection tract entering the cerebralcortex and extending through the corpus callosum to reach theSVZa. The GFP–GRP cells were observed along the full extent ofthe RMS, including the SVZa and the subependymal zone in themiddle of the OB as well as in the overlying granule cell layer.Relatively few GFP–GRP cells departed from the RMS beforeentering the OB. The majority of the cells were situated along theanterior–posterior axis of the corpus callosum and in the overlyingsubplate and layer VI of the occipital cortex. A similar pattern ofdistribution was seen at 7 and 14 days after transplantation.(AOB, accessory olfactory bulb; CC, corpus callosum; epl, externalplexiform layer; gcl, granule cell layer; gl, glomerular layer; HP,hippocampus; LV, lateral ventricle; mcl, mitral cell layer; OB,olfactory bulb; RMS, rostral migratory stream; SEZ, subependy-mal zone; SVZa, anterior subventricular zone; SVZp posteriorsubventricular zone.)

15IN VIVO CHARACTERIZATION OF GLIAL-RESTRICTED PRECURSORS

GFP-expressing cells and a rhodamine-conjugatedsecondary antibody to determine the antigenic pheno-type.

Adult sections were immunostained after 7 and 28days. Transplanted and control rats were anesthetizedand perfused transcardially with 4% PFA for 5–8 min.Brains were removed and fixed in 4% PFA for 4 h.Fifty-micrometer sections were cut on a Vibratome andwere incubated for 1 h at RT in blocking solution (4.0%NGS and 0.3% TritonX-100 in PBS (pH 7.2) prior tostaining. The following primary antibodies were used:rabbit polyclonal anti-GFAP (Dako, 1:1200), mouseanti-S100 (Chemicon 1:1000) used to identify astrocytes,and mouse anti-neurofilament M (Chemicon, 1:1000)or mouse anti-b-type III tubulin (Southern Biotechnol-ogy 1:1500) to identify neurons. All antibodies werediluted with the blocking solution. Secondary antibod-ies, used at a dilution of 1:200, were goat anti-mouseIgG (H1L) Alexa 564 (Molecular Probes) and goat anti-rabbit Ig (H1L) Alexa 594 (Molecular Probes). Sectionswere mounted onto slides using Fluoromount (Molecu-lar Probes) and viewed using a Nikon immunofluores-cence microscope. Images were captured using a Cool-snap digital camera (S&M microscopes).

RESULTS

GFP Expression in GRP Cells Does Not Alter TheirDifferentiation Potential

To be able to distinguish graft from host cells wepurified GRP cells and infected the pure populationwith a GFP-expressing retrovirus. The relatively smallnumber of GFP-expressing cells was isolated by FACScell sorting and positive cells were expanded for 2weeks in culture. This procedure results in an 80–100% GFP-expressing cell population that can easilybe detected posttransplantation. To ensure that theinfected cells did not change their biological properties,we plated GFP-expressing cells in oligodendrocyte orastrocyte inducing condition and stained the cells after7 days in culture. As shown in Figs. 1A–1C, GFP-expressing cells exposed to bFGF/PDGF and thyroidhormone (T3) differentiate into GalC-immunoreactiveoligodendrocytes, while GFP-expressing cells plated inBMP-4-containing medium differentiate into GFAP-expressing astrocytes. We furthermore did not observeany increased cell death or cell division in GFP-ex-pressing cultures compared to non-GFP-expressingcells (data not shown). Both control and GFP-express-ing cells were plated at the same density and the totalnumber of cells was counted at the end of the experi-ment using DAPI staining. We could not detect anysignificant difference in the total number of DAPI1

cells.

GRP Cells Transplanted to a Neonatal NeurogenicRegion Do Not Generate Neurons but Differentiateinto Astrocytes and Oligodendrocytes

GRP cells were transplanted into the anterior SVZ ofneonatal animals, a region that has been shown toprovide an environment that allows neuronal differen-tiation from neuron competent precursor cells (3, 5, 22,36, 51). After 3, 7, and 14 days animals were sacrificedand double-label immunocytochemistry was performedusing antibodies against the astrocyte marker GFAP,the oligodendrocyte marker proteolipid protein (PLP-DM20), and the neuronal marker type III b-tubulinalong with anti-GFP to enhance recognition of thetransplanted GRP cells and their progeny. The resultsare summarized in Table 1 and representative imagesare shown in Fig. 2.

In none of the regions and at no time points were theGFP–GRP cells found to express type III b-tubulin(Fig. 2C). Three days after transplantation, however,the majority of the transplanted GRP cells expressedGFAP (Fig. 2B), while very few cells expressed pro-teolipid protein (PLP/DM20). At 7 and 14 days aftertransplantation more GRP-derived cells were PLP/DM20 positive (Figs. 2D and 2E) although the cells didnot yet show myelin elaborations, which is a phenotypeconsistent with newly forming oligodendrocytes (46).Taken together these findings show that similar totheir properties in vitro, A2B5-positive GRP cells dif-ferentiated into astrocytes and expressed an earlymarker of oligodendrocytes in vivo but did not generateneurons, demonstrating their commitment to the gliallineage.

GRP Cells Migrate Extensively in the Neonatal Brain

In addition to the differentiation profile of the trans-planted cells we also determined their ability to mi-grate in the neonatal host brain. We found that GFP–GRP cells migrated extensively in both the rostral andthe caudal direction in the host brain. Figure 3 depictsa line drawing of a sagittal view of the rat brain show-ing the regions to which the transplanted GFP–GRPcells had migrated. In the rostral direction, graftedcells migrated along the rostral migratory stream(RMS) to the olfactory bulb (OB), similar to endogenousneuronal restricted progenitor cells from the SVZa (23,50). The cells were confined to the RMS while en routeto the OB unlike NRP cells from the embryonic spinalcord, which when transplanted into the SVZa, enteredthe surrounding anterior olfactory nuclei (Luskin et al.,unpublished observation). Once the GRP cells reachedthe rostral tip of the RMS, corresponding to the sub-ependymal zone (SEZ) of the OB, the cells entered theoverlying granule cell layer, but did not migrate fur-ther outward in the bulb at any of the posttransplanttimes examined.

oaadG

16 HERRERA ET AL.

The majority of the transplanted cells, however, mi-grated rostrally and caudally in the corpus callosumand to the subventricular zone posterior to the SVZa(the SVZp)—two highly gliogenic regions in the neona-tal brain. The overlying cortex was largely spared ex-

FIG. 4. GRP cells express astrocytic markers after transplantatif transplantation. B and D, GFP-expressing GRP cells that were trnti-S-100 (A) or anti-GFAP antibody (C and E) in red. GFP-expresslso labeled after 28 days with anti-GFAP antibody (E). A substantifferentiate into GFAP-immunoreactive cells that are interspersedFP-expressing graft-derived cells that also express GFAP. Bar, 20

cept for layer VI, where a small number of cells werevisualized. The cells did not migrate to regions adja-cent to the lateral ventricle and the SVZa, such as thehippocampus or the striatum. With the exception of theRMS, and a small number of cells in layer VI of the

nto the adult CNS. Shown are representative sections after 28 daysplanted into the adult corpus callosum. Sections were stained withcells transplanted into the adult SVZ are shown in F. Sections werenumber of the transplanted cells (that are initially GFAP negative)th GFAP-immunoreactive negative cells. Arrows highlight some of.

on iansingial

wimm

17IN VIVO CHARACTERIZATION OF GLIAL-RESTRICTED PRECURSORS

cortex, the transplanted GFP–GRP cells migrated intogliogenic sites.

Transplanted GRP Cells Generate Astrocytesin the Adult Brain

To determine that the ability of transplanted GRPcells to generate astrocytes was not limited to neonataltransplants, we next grafted cells into SVZ and corpus

FIG. 5. GRP cells do not express neuronal markers following ttransplanted into the adult SVZa was analyzed using antibodies agvisualized with a dual FITC/rhodamine filter 1 week posttranspposttransplantation. None of the GFP-expressing cells labeled with aamplified in the adult transplants. Bar, 20 mm.

FIG. 6. Myelination in the md rat after GRP cell transplantattransplanted into the dorsal column of 7-day-old md rats. After 2 wand C) A representative section of a nontransplanted animal stainedA representative section of a transplanted spinal cord stained with

callosum of adult rats. After 7 and 28 days animalswere sacrificed and sections were analyzed. At bothtime points we were able to detect GFP-expressinggraft cells in both the corpus callosum and the SVZ.Using immunostaining with anti-GFAP and anti-S-100, we determined that graft-derived cells expressedboth of these astrocytic markers already after 1 week.Cells showed clearly multiple processes with an aver-

splantation into the adult SVZ. The phenotype of GFP–GRP cellsst neurofilament-M. (A) A representative section of the adult SVZtation. (B) A representative section of the adult SVZa 28 daysodies against neurofilament-M. Note that the GFP expression is not

GRP cells were isolated and purified from E13.5 spinal cord ands following transplantation, 5-mm sections were immunostained. (Ah anti-PLP (upper level) and toluidine blue (lower levels). (B and D)i-PLP (upper level) and toluidine blue (lower levels). Bar, 25 mm.

ranainlanntib

ion.eekwitant

A

vpfo

18 HERRERA ET AL.

age process length of 25–32 mm. After 4 weeks GFAPand S-100 labeling was still prominent in the trans-planted GFP-expressing cells (Fig. 4). Sections of ani-mals that received transplants into the SVZ were alsoexamined with antibodies against b-III tubulin andneurofilament-M after 7 and 28 days. While endoge-nous neurons strongly labeled with anti-b-III tubulin(data not show) or neurofilament-M, none of the trans-planted GFP-expressing GRP cells were immunoreac-tive against these markers (Fig. 5).

Transplanted GRP Cells Generate Myelin-ProducingOligodendrocytes in the Neonatal Spinal Cord

To establish whether GRP cells have the ability invivo to generate myelin-producing oligodendrocytes,GRP cells were isolated and purified from E13.5 spinalcord and transplanted into the dorsal column of 7-day-old md rats. Two weeks following transplantation, allgrafted rats showed a white patch (gross appearance ofmyelin) in the dorsal spinal cord. The length of thewhite patch along the dorsal column varied but was aslong as 5 mm. Immunostaining of the grafted spinalcord indicated that numerous myelin sheaths, predom-inantly in the dorsal funiculus, were positive for PLP,whereas the untransplanted cord was devoid of PLP(Fig. 6). The presence of transplant-derived myelinsheaths was confirmed in toluidine blue-stained 1-mmsections showing many myelinated axons along witholigodendrocytes (Fig. 6). All nine md rats that re-ceived grafts showed a consistent generation of myelin-producing cells from the grafted GRP cells.

DISCUSSION

The identification of GRP cells and their extensive invitro characterization has led to the question ofwhether the different properties of this cell type areretained in a complex in vivo environment. The ques-tions we specifically wanted to answer were whetherGRP cells are glial-restricted precursor cells that cangenerate both oligodendrocytes and astrocytes in vivo.

strocytic Differentiation in Vitro and in Vivo

One of the most intriguing properties of GRP cells initro is their ability to generate different astrocytichenotypes (33). This is a very different observationrom that obtained with O-2A progenitor cells, whichnly generate one kind of astrocyte in vitro. The differ-

ence in the ability to astrocytic differentiation is evenmore prominent in vivo. It has been reported thatprimary O-2A cells when transplanted into the neona-tal brain fail to generate astrocytes (4, 10, 17), al-though some glial progenitor cell lines seem to have thecapacity to generate astrocytes in vivo GRP cells (1,13). This observation could be interesting as it mightsuggest that the cell line in fact represents more the

GRP cell type than the O-2A/OPC cell types enablingthem to generate astrocytes in vivo. In respect to pri-mary cells it seems clear that the generation of astro-cytes from GRP cells in vivo is a unique property ofthese cells that is not shared with primary O-2A/OPCcells. Our results further show that the generation ofastrocytes from transplanted GRP cells is not re-stricted to the neonatal brain but also occurs in theadult brain. GFAP-expressing cells can be found inboth animal groups as early as 7 days after transplan-tation. In the adult the appearance of GRP-cell-derivedastrocytes is most apparent in the corpus callosumwhere clearly defined graft derived astrocytes linealong the corpus callosum. While we see GFP labeledastrocytes in the corpus callosum spreading as far as100 mm from the injection site we cannot distinguishwith confidence whether this spreading is a result ofmigration or is a consequence of the injection itself.Experiments analyzing later time points are underway and will clarify this issue.

While graft-derived astrocytes show similar anti-genic characteristics independent of their site of ap-pearance (CC or SVZ in the adult brain), we coulddetect a striking difference in the morphology (as mea-sured in process length) of the generated astrocytesover time posttransplantation (data not shown). Thefull implication of this finding is at the moment notclear. A variety of reports suggest that morphologicalchanges in astrocytes might be an evidence for differ-ent functional properties (reviewed by 43). A recentstudy has linked morphological differences on astro-cytes with the differential expression of the GLT-1 andGLAST glutamate transporters (31). The same inves-tigators show that this differential expression was cor-related with the maturation of the cultures suggestingan active role of the microenvironment in the functionand maturation of astrocytes.

Lineage Restriction

Our initial definition of the lineage restriction ofGRP cells to glial phenotypes was based on in vitroexperiments in which we exposed GRP cells to cultureconditions that induce the generation of neurons fromtotipotent neuroepithelial stem cells and from neuron-restricted precursor cells and then determined the abil-ity of the GRP cells to generate neurons. We now haveextended these in vitro results with in vivo observa-tions. The transplantation of GRP cells into the SVZregion yielded no graft-derived cells that expressedneuronal markers, even when cells migrated alongpathways, such as the rostral migratory stream, inwhich neuronal generation occurs readily followingtransplantation of NEP and NRP cells.

The failure of GRP cells to generate neurons may beof particular interest in light of recent data by Kondoand Raff (19) who suggest that under certain in vitro

mtmsfibttstid

M

rtismpencvGstems

dsnidiOt

O

igtGeoc(

ciceictTleettamciisobtateaimotwTnwt

gtGtca

19IN VIVO CHARACTERIZATION OF GLIAL-RESTRICTED PRECURSORS

conditions O-2A/OPC cells can be forced to differenti-ate into neurons. Our data show, however, that GRPcells do not generate neurons even when they are ex-posed to the most complex neurogenic in vivo environ-

ent in which neuron competent cells readily differen-iate into neurons (11, 16, 25, 34, 42). This result couldean that either GRP cells are terminally lineage re-

tricted, that “reprogramming” events (terminology de-ned by Kondo and Raff) play no role in the normalrain or that cultures studied by Kondo and Raff con-ained a small number of stem cells that were inducedo proliferate. Such a possibility has been discussedome time ago by Omlin and Waldmeyer who describehe presence of a population of “undifferentiated” cellsn optic nerve derived long-term cultures that seem toifferentiate into neuron-like cells (29).

igration

In addition to the demonstration that GRP cells areestricted to the glial lineage, the transplantation intohe neonatal brain yielded other significant insightsnto the biology of these cells. GRP cells showed exten-ive migration in the neonatal brain along the rostraligratory stream. It was interesting to note that the

rogeny of the transplanted GRP cells were found pref-rentially in regions of the CNS in which predomi-antly glial cells are generated, such as the corpusallosum. We do not know whether this distribution inivo reflects a preferential migration or a failure ofRP cells to survive in neurogenic regions. These re-

ults do, however, reveal a striking difference betweenhe behavior of these cells and of the totipotent neuro-pithelial stem cells that have been transplanted byultiple laboratories in previous studies (for review

ee 34).Transplants into the adult brain yielded different

egrees of migration depending on the implantationite. While SVZ transplants can be found predomi-antly close to the implantation site, cells transplanted

nto the corpus callosum seem to display a certainegree of migration along the white matter tract. Thiss of particular interest as it has been shown that-2A/OPC cells survive poorly and hardly migrate in

he adult brain (28).

ligodendrocytic Differentiation

Our data show that transplanted GRP cells surviven the dysmyelinated rat spinal cord and produce oli-odendrocytes that contribute to myelination of ex-ended regions of the cord. The extent to which graftedRP cells migrate along the dorsal funiculus and my-linate axons is similar to that produced by graftedligodendroglial progenitors derived from neural stemells (49) and the grafted CG4 oligodendrocyte cell line40).

The differentiation of GRP cells into oligodendro-ytes seems not to be restricted to the transplantationnto a mutant environment, where the behavior of graftells might be different from that seen in a nonmutantnvironment (12, 28). The transplantation of GRP cellsnto the normal neonatal brain yielded graft-derivedells that express PLP/DM20 protein suggesting thathese cells are differentiating into oligodendrocytes.he expression of PLP and its isoform DM20 has been

inked to the commitment of precursor cells to differ-ntiate into oligodendrocytes and the absence of PLP-xpressing cells is associated with a defect in myelina-ion (6, 8, 24, 27, 35, 38, 39, 47). The result indicateshat GRP cells do not only generate oligodendrocytes in

mutant environment like that of the md rat, butight also participate in the normal myelination pro-

ess that is in the neonate still in progress. Interest-ngly, we did not observe differentiation of graft cellsnto oligodendrocytes in the normal adult corpus callo-um (in contrast to the neonatal brain). This inhibitionf oligodendrocyte differentiation in the normal adultrain is consistent with the observation showing thaterminal differentiated oligodendrocytes can survivend remyelinate normal brain regions, while progeni-or populations consistently fail to initiate the differ-ntiation process into oligodendrocytes in a normaldult environment (20, 44). The outcome of transplant-ng glial precursor cells into an normal adult environ-

ent seems to depend on the differentiation potentialf the graft cells: O-2A/OPC cells that are committed tohe oligodendrocyte lineage die in this environmenthile less restricted GRP cells generate astrocytes.his observation suggests that both environment sig-als and the competence of the cell receiving the signalill ultimately determine the outcome of transplanta-

ion experiments.In summary, our findings show that GRP cells do not

enerate neurons in vivo confirming their restriction tohe glial lineage we had characterized in vitro. WhileRP cells, like many other glial precursor cells, have

he capacity to generate myelin-producing oligodendro-ytes in vivo, they are in addition able to generatestrocytes in the neonatal and adult brain.

ACKNOWLEDGMENTS

We thank Professor Dr. Mark Noble and Dr. Mahendra Rao forcritical comments and suggestions, Darin Messina for technical ad-vice, and Samuel Bernard for excellent technical assistance. Thework was supported by: M.M.P., J.H., and C.P., the HuntsmanCancer Institute; M.M.P., the National Multiple Sclerosis Society;M.M.P. and P.T., the Keck Center for Tissue Engineering; I.D.D.,Grant NS 33710/NIH; the Myelin Project; and the Oscar RennebohmFoundation.

REFERENCES

1. Barnett, S. C., R. J. Franklin, and W. F. Blakemore. 1993. Invitro and in vivo analysis of a rat bipotential O-2A progenitor

1

1

1

1

1

1

1

1

1

1

2

2

2

2

2

2

3

3

3

3

3

20 HERRERA ET AL.

cell line containing the temperature-sensitive mutant gene ofthe SV40 large T antigen. Eur. J. Neurosci. 5: 1247–1260.

2. Bignami, A., L. F. Eng, D. Dahl, and C. T. Uyeda. 1972. Local-ization of the glial fibrillary acidic protein in astrocytes byimmunofluorescence. Brain Res. 43: 429–435.

3. Calof, A. L., J. S. Mumm, P. C. Rim, and J. Shou. 1998. Theneuronal stem cell of the olfactory epithelium. J. Neurobiol. 36:190–205.

4. Crang, A. J., R. J. Franklin, W. F. Blakemore, M. Noble, S. C.Barnett, A. Groves, J. Trotter, and M. Schachner. 1992. Thedifferentiation of glial cell progenitor populations followingtransplantation into non-repairing central nervous system gliallesions in adult animals. J. Neuroimmunol. 40: 243–253.

5. Doucette, J. R., J. A. Kiernan, and B. A. Flumerfelt. 1983. There-innervation of olfactory glomeruli following transection ofprimary olfactory axons in the central or peripheral nervoussystem. J. Anat. 137(Pt 1): 1–19.

6. Duchala, C. S., K. Asotra, and W. B. Macklin. 1995. Expressionof cell surface markers and myelin proteins in cultured oligo-dendrocytes from neonatal brain of rat and mouse: A compara-tive study. Dev. Neurosci. 17: 70–80.

7. Duncan, I. D. 1996. Glial cell transplantation and remyelina-tion of the central nervous system. Neuropathol. Appl. Neuro-biol. 22: 87–100.

8. Duncan, I. D., J. P. Hammang, S. Goda, and R. H. Quarles.1989. Myelination in the jimpy mouse in the absence of prote-olipid protein. Glia 2: 148–154.

9. Eisenbarth, G. S., F. S. Walsh, and M. Nirenberg. 1979. Mono-clonal antibody to plasma membrane antigen of neurons. Proc.Natl. Acad. Sci. USA 76: 4913–4917.

0. Espinosa De Los Monteros, A., M. Zhang, and J. De Vellis.1993. O2A progenitor cells transplanted into the neonatal ratbrain develop into oligodendrocytes but not astrocytes. Proc.Natl. Acad. Sci. USA 90: 50–54.

1. Fisher, L. J. 1997. Neural precursor cells: Applications for thestudy and repair of the central nervous system. Neurobiol. Dis.4: 1–22.

2. Franklin, R. J., S. A. Bayley, and W. F. Blakemore. 1996.Transplanted CG4 cells (an oligodendrocyte progenitor cell line)survive, migrate, and contribute to repair of areas of demyeli-nation in X-irradiated and damaged spinal cord but not innormal spinal cord. Exp. Neurol. 137: 263–276.

3. Franklin, R. J., S. A. Bayley, R. Milner, C. ffrench-Constant,and W. F. Blakemore. 1995. Differentiation of the O-2A progen-itor cell line CG-4 into oligodendrocytes and astrocytes follow-ing transplantation into glia-deficient areas of CNS white mat-ter. Glia 13: 39–44.

4. Franklin, R. J., and W. F. Blakemore. 1995. Glial-cell trans-plantation and plasticity in the O-2A lineage—Implications forCNS repair. Trends Neurosci. 18: 151–156.

5. Gard, A. L., W. C. N. Williams, and M. R. Burrell. 1995. Oligo-dendroblasts distinguished from O-2A glial progenitors by sur-face phenotype (O41GalC2) and response to cytokines usingsignal transducer LIFR beta. Dev. Biol. 167: 596–608.

6. Gritti, A., L. Cova, E. A. Parati, R. Galli, and A. L. Vescovi.1995. Basic fibroblast growth factor supports the proliferationof epidermal growth factor-generated neuronal precursor cellsof the adult mouse CNS. Neurosci. Lett. 185: 151–154.

7. Groves, A. K., S. C. Barnett, R. J. Franklin, A. J. Crang, M.Mayer, W. F. Blakemore, and M. Noble. 1993. Repair of demy-elinated lesions by transplantation of purified O-2A progenitorcells. Nature 362: 453–455.

8. Koeppen, A. H., K. D. Barron, C. K. Csiza, and E. A. Greenfield.A. 1988. Comparative immunocytochemistry of Pelizaeus-

Merzbacher disease, the jimpy mouse, and the myelin-deficientrat. J. Neurol. Sci. 84: 315–327.

9. Kondo, T., and M. Raff. 2000. Oligodendrocyte precursor cellsreprogrammed to become multipotential CNS stem cells [seecomments]. Science 289: 1754–1757.

20. Lachapelle, F., E. Duhamel Clerin, A. Gansmuller, A. BaronVan Evercooren, H. Villarroya, and M. Gumpel. 1994. Trans-planted transgenically marked oligodendrocytes survive, mi-grate and myelinate in the normal mouse brain as they do inthe shiverer mouse brain. Eur. J. Neurosci. 6: 814–824.

21. Law, A. K., V. Pencea, C. R. Buck, and M. B. Luskin. 1999.Neurogenesis and neuronal migration in the neonatal rat fore-brain anterior subventricular zone do not require GFAP-posi-tive astrocytes. Dev. Biol. 216: 622–634.

22. Luskin, M. B. 1994. Neuronal cell lineage in the vertebratecentral nervous system. FASEB J. 8: 722–730.

23. Luskin, M. B., J. G. Parnavelas, and J. A. Barfield. 1993.Neurons, astrocytes, and oligodendrocytes of the rat cerebralcortex originate from separate progenitor cells: An ultrastruc-tural analysis of clonally related cells. J. Neurosci. 13: 1730–1750.

4. Macklin, W. B., M. V. Gardinier, Z. O. Obeso, K. D. King, andP. A. Wight. 1991. Mutations in the myelin proteolipid proteingene alter oligodendrocyte gene expression in jimpy and jimpy-msd mice. J. Neurochem. 56: 163–171.

5. Marvin, M., and R. Mckay. 1992. Multipotential stem cells inthe vertebrate CNS. Semin. Cell Biol. 3: 401–411.

6. Mi, H., and B. Barres. 1999. Purification and characterizationof astrocyte precursor cells in the developing rat optic nerve.J. Neurosci. 19: 1049–1061.

7. Nadon, N. L., and I. D. Duncan. 1995. Gene expression andoligodendrocyte development in the myelin deficient rat.J. Neurosci. Res. 41: 96–104.

8. O’leary, M. T., and W. F. Blakemore. 1997. Oligodendrocyteprecursors survive poorly and do not migrate following trans-plantation into the normal adult central nervous system.J. Neurosci. Res. 48: 159–167.

9. Omlin, F. X., and J. Waldmeyer. 1989. Differentiation of neu-ron-like cells in cultured rat optic nerves: A neuron or commonneuron-glia progenitor? Dev. Biol. 133: 247–253.

0. Pear, W. S., G. P. Nolan, M. L. Scott, and D. Baltimore. 1993.Production of high titer retrovirus by transient transfection.Proc. Natl. Acad. Sci. USA 90: 8392–8396.

1. Perego, C., C. Vanoni, M. Bossi, S. Massari, H. Basudev, R.Longhi, and G. Pietrini. 2000. The GLT-1 and GLAST gluta-mate transporters are expressed on morphologically distinctastrocytes and regulated by neuronal activity in primary hip-pocampal cocultures. J. Neurochem. 75: 1076–1084.

2. Rao, M., and M. Mayer-Proschel. 1997. Glial restricted precur-sors are derived from multipotent neuroepithelial stem cells.Dev. Biol. 188: 48–63.

3. Rao, M., M. Noble, and M. Mayer-Proschel. 1998. A tripotentialglial precursor cell is present in the developing spinal cord.Proc. Natl. Acad. Sci. USA 95: 3996–4001.

4. Rao, M. S. 1999. Multipotent and restricted precursors in thecentral nervous system. Anat. Rec. 257: 137–148.

35. Redwine, J. M., and R. C. Armstrong. 1998. In vivo proliferationof oligodendrocyte progenitors expressing PDGFalphaR duringearly remyelination. J. Neurobiol. 37: 413–428.

36. Scott, D. E., and S. L. Hansen. 1997. Post-traumatic regenera-tion, neurogenesis and neuronal migration in the adult mam-malian brain. Virginia Med. Q. 124: 249–261.

21IN VIVO CHARACTERIZATION OF GLIAL-RESTRICTED PRECURSORS

37. Seidman, K. J., A. L. Teng, R. Rosenkopf, P. Spilotro, and J. A.Weyhenmeyer. 1997. Isolation, cloning and characterization ofa putative type-1 astrocyte cell line. Brain Res. 753: 18–26.

38. Spassky, N., C. Goujet-Zalc, E. Parmantier, C. Olivier, S. Mar-tinez, A. Ivanova, K. Ikenaka, W. Macklin, I. Cerruti, B. Zalc,and J. L. Thomas. 1998. Multiple restricted origin of oligoden-drocytes. J. Neurosci. 18: 8331–8343.

39. Timsit, S., S. Martinez, B. Allinquant, F. Peyron, L. Puelles,and B. Zalc. 1995. Oligodendrocytes originate in a restrictedzone of the embryonic ventral neural tube defined by DM-20mRNA expression. J. Neurosci. 15: 1012–1024.

40. Tontsch, U., D. R. Archer, M. Dubois-Dalcq, and I. D. Duncan.1994. Transplantation of an oligodendrocyte cell line leading toextensive myelination. Proc. Natl. Acad. Sci. USA 11616–11620.

41. Trotter, J., A. J. Crang, M. Schachner, and W. F. Blakemore.1993. Lines of glial precursor cells immortalised with a temper-ature-sensitive oncogene give rise to astrocytes and oligoden-drocytes following transplantation into demyelinated lesions inthe central nervous system. Glia 9: 25–40.

42. Vescovi, A. L., E. A. Parati, A. Gritti, P. Poulin, M. Ferrario, E.Wanke, P. Frolichsthal-Schoeller, L. Cova, M. Arcellana-Pan-lilio, A. Colombo, and R. Galli. 1999. Isolation and cloning ofmultipotential stem cells from the embryonic human CNS andestablishment of transplantable human neural stem cell linesby epigenetic stimulation. Exp. Neurol. 156: 71–83.

43. Walz, W. 2000. Controversy surrounding the existence of dis-crete functional classes of astrocytes in adult gray matter. Glia31: 95–103.

44. Warrington, A. E., E. Barbarese, and S. E. Pfeiffer. 1993. Dif-ferential myelinogenic capacity of specific developmental stages

of the oligodendrocyte lineage upon transplantation into hypo-myelinating hosts. J. Neurosci. Res. 34: 1–13.

45. Weimbs, T., T. Dick, W. Stoffel, and E. Boltshauser. 1990. Apoint mutation at the X-chromosomal proteolipid protein locusin Pelizaeus-Merzbacher disease leads to disruption of myeli-nogenesis. Biol. Chem. Hoppe Seyler 371: 1175–1183.

46. Yang, X., and R. P. Skoff. 1997. Proteolipid protein regulatesthe survival and differentiation of oligodendrocytes. J. Neuro-sci. 17: 2056–2070.

47. Yu, W. P., E. J. Collarini, N. P. Pringle, and W. D. Richardson.1994. Embryonic expression of myelin genes: Evidence for afocal source of oligodendrocyte precursors in the ventricularzone of the neural tube. Neuron 12: 1353–1362.

48. Zhang, S. C., B. Ge, and I. D. Duncan. 1999. Adult brain retainsthe potential to generate oligodendroglial progenitors with ex-tensive myelination capacity. Proc. Natl. Acad. Sci. USA 96:4089–4094.

49. Zhang, S. C., C. Lundberg, D. Lipsitz, L. T. O’Connor, and I. D.Duncan. 1998. Generation of oligodendroglia progenitors fromneural stem cells. J. Neurocytol. 27: 475–489.

50. Zigova, T., R. Betarbet, B. J. Soteres, S. Brock, R. A. Bakay, andM. B. Luskin. 1996. A comparison of the patterns of migrationand the destinations of homotopically transplanted neonatalsubventricular zone cells and heterotopically transplanted tel-encephalic ventricular zone cells. Dev. Biol. 173: 459–474.

51. Zigova, T., V. Pencea, R. Betarbet, S. J. Wiegand, C. Alexander,R. A. Bakay, and M. B. Luskin. 1998. Neuronal progenitor cellsof the neonatal subventricular zone differentiate and dispersefollowing transplantation into the adult rat striatum. CellTransplant. 7: 137–156.