haematopoietic progenitor cells from adult bone marrow differentiate into cells that express...

SHORT COMMUNICATIONHaematopoietic progenitor cells from adult bone marrowdifferentiate into cells that express oligodendroglialantigens in the neonatal mouse brain

Sonia Bonilla, Pedro AlarcoÂn, RamoÂn Villaverde,1 Pedro Aparicio,2 Augusto Silva3 and Salvador MartõÂnezInstituto de Neurociencias, UMH-CSIC, Campus San Juan, Alicante, Spain1Hospital General Universitario. Servicio de NeurologõÂa, Universidad de Murcia, Murcia, Spain2Dpto. BioquõÂmica B. InmunologõÂa. Universidad de Murcia, Murcia, Spain3Centro de Investigaciones BioloÂgicas, Madrid, Spain

Keywords: cell grafts, cell sorting, haematopoietic progenitors, Multiple Sclerosis, oligodendrocytes, remyelination, stem cells

Abstract

Stem cells are self-renewable, pluripotent cells that, in adult life, proliferate by a characteristic asymmetric division in which one

daughter cell is committed to differentiation whereas the other remains a stem cell. These cells are also characterized by their

ability to differentiate into various cell types under heterotopic environmental in¯uences. In the present study, we have explored

the potential of adult haematopoietic bone marrow cells to differentiate into cells of oligodendroglial lineage under physiological,active myelinating conditions. We present evidence of generation of cells expressing oligodendroglial speci®c markers from a

bone marrow subpopulation enriched on adult haematopoietic progenitor cells (CD117+) in vivo after intracerebral transplantation

into the neonatal mouse brain. Our results suggest that adult bone marrow cells have the capacity to undergo differentiation fromhaematopoietic to oligodendroglial cells and add support the validity of bone marrow transplants as an alternative treatment for

demyelinating diseases of the CNS including Multiple Sclerosis.

Introduction

Stem cells are self-renewable, pluripotent cells that, in adult life,

proliferate by a characteristic asymmetric division whereby one

daughter cell is committed to differentiation while the other remains

undifferentiated. Differences in stem cell types depend on their

localization and differentiation potential (Fuchs & Segre, 2000;

Weissman, 2000) and they can be transformed into heterogenic cell

types by means of ectopic in¯uences (Alison et al. 2000; Clarke et al.

2000; Blau et al. 2001). In adult bone marrow, haematopoietic

(HSCs) and stromal (MSCs) stem cells are present (Weissman, 2000).

In relation to the central nervous system (CNS), mouse bone

marrow cells (BMCs) or selected mouse and human MSCs are

capable of being incorporated into the CNS and then to differentiate

into neurons, astrocytes and microglia (Eglitis & Mezey, 1997; Azizi

et al., 1998; Kopen et al., 1999; Brazelton et al. 2000; Mezey et al.

2000; reviewed in, Blau et al. 2001). Recently, Sasaki et al. (2001)

demonstrated the differentiation of BMCs into oligodendrocytes after

they were grafted into irradiated rat spinal cord. In addition, human

MSCs have been reported to develop a neuronal phenotype in vitro

(Woodbury et al., 2000). Moreover, neural stem cells can generate

HSCs in vivo and restore blood progenitors (Bjornson et al., 1999).

Nevertheless, the potential of HSCs to develop into oligodendrocytes

and other neural cells in vivo, has not yet been speci®cally explored in

detail.

The present work studied the potential of HSCs to differentiate into

oligodendrocyte progenitors under physiological, active myelinating

conditions, such as those that are present in the neonatal brain. To this

purpose, we selected BMC subpopulations enriched with, or deprived

of CD117+ cells. These cells were identi®ed by the expression of c-

kit (CD117), a marker of HSCs. The possible oligodendroglial

potential of HSCs could render these cells useful as a component of

cellular therapy for human demyelinating diseases, such as Multiple

Sclerosis (MS). Indeed, bone marrow grafts have already been used in

several clinical MS trials (reviewed in, Burns & Burt, 1999; Saiz et

al. 2001).

Multiple Sclerosis is an in¯ammatory, autoimmune disease of the

CNS, involving disseminate plaque formation. At the initial stages of

demyelinization, oligodendrocyte progenitors reactivate the remyeli-

nating process (Allen & Kirk, 1997; BruÈck et al. 2001) but when the

regenerative attempt fails to restore the myelin sheet, the associated

plaques progress to form a chronic lesion. The possible use of cell

grafts in MS therapeutic trials was initially limited by the disseminate

characteristic of the disease. However, the proliferative and migratory

abilities of oligodendrocyte progenitors in the CNS (Archer et al.,

1997; Franklin & Blakemore, 1997; Ader et al. 2000), together with

oligodendrocyte generation from BMCs (Sasaki et al. 2001) and

HSCs (the present work), provide the possibility that HSCs constitute

a viable alternative therapy for MS.

In order to unequivocally identify donor cells in the transplanted

host, we employed two strategies: (i) HSCs cells were obtained from

transgenic mice which have the b-gal gene under the control of the

Correspondence: Dr Salvador MartõÂnez., Instituto de Neurociencias, as above.E-mail: [email protected]

Received 27 April 2001, revised 31 December 2001, accepted 3 January 2002

European Journal of Neuroscience, Vol. 15, pp. 575±582, 2002 ã Federation of European Neuroscience Societies

plp/dm20 gene promoter (plp-sh ble-LacZ mice; Spassky et al.,

1998), thus permitting an identi®cation of donor cells with an

oligodendroglial phenotype by the presence of X-gal cytoplasm

deposits and (ii) the use of host and donor mice expressing a different

H-2 mayor histocompatibility complex. We use H-2k mice to graft

HCSs cells derived from H-2d mice which allows the identi®cation of

the host cells by immunolabelling using antibodies to H-2d antigen.

We report here, for the ®rst time, the possibility to generate cells

expressing speci®c oligodendroglial markers from adult HSCs. Our

results support the validity of bone marrow stem cell grafts as an

alternative regenerative therapy for demyelinating diseases of the

CNS including MS.

Materials and methods

Isolation of bone marrow cells and CD117+ cell enrichment

All experiments were carried out according to the guidelines laid

down by the European Council Directive for the Care of Laboratory

Animals. We obtained HSCs from the bone marrow of sh ble-LacZ

transgenic mice which contain the sh ble-LacZ fusion gene driven by

the promoter and regulatory sequences of the plp gene (Spassky et al.,

1998), encoding proteolipid protein, the major protein of CNS

myelin. Brie¯y, BMCs were obtained from the femurs of adult

transgenic mice, anaesthetized with an overdose of chloroform, by

¯ushing out the bone marrow with complete RPMI 1640 medium

(Gibco BRL, Life technologies, Paisley, Scotland) containing 10%

fetal calf serum (FCS, BioWhittaker, Cambrex, NY) and 1 mM

glutamine (Gibco BRL, Life technologies, Paisley, Scotland). Bone

marrow cells from 10 adult mice were treated with 5 mL of 0.144 M

ammonium chloride in 17 mM Tris-HCl, pH 7.2, for 5 min at RT to

remove erythrocyte contamination. A sample of BMCs was stained

with phycoerythrin labelled CD117 (CD117-PE) and ¯uorescein

labelled Sca-1 (Sca-1-FITC) antibodies (BD Pharmingen, San Diego,

CA) in order to localize the CD117+/Sca-1+ subpopulation on the

FSC/SSC dotblot. Bone marrow cells were resuspended in RPMI

medium without FCS at 2 3 106 cells/mL and sorted in a Vantage

Fluorescence Activated Cell Sorter (FACS; Becton Dickinson, USA)

equipped with an argon-ion laser, tuned at an excitation wavelength

of 488 nm. Two different subpopulations of bone marrow cells were

gated and collected according to the forward (FSC) and side scattered

(SSC) signals (cell complexity and size, respectively: R1 and R2;

Fig. 1A). The R2 subpopulation was enriched in c-kit+ cells (» 20%),

whereas these cells were scarce in the R1 subpopulation (< 3%).

FIG. 1. Experimental procedures. (A) Two different bone marrow subpopulations (R1 and R2, green and red squares, respectively) from plp-sh ble-LacZtransgenic mice were gated in the sorting protocol and grafted into the brain of neonatal (P0) mice. The R2 subpopulation was selected as an experimentalcell population, containing an enriched population of c-kit+ (CD117+) cells (around 20%) in which both primitive haematopoietic progenitors (CD117+, Sca-1+) and more committed haematopoietic progenitors (CD117+, Sca-1±) are represented. The R1 subpopulation was considered as a control population as mostof the cells are lymphocytes (CD117±, Sca-1+). (B) Bone marrow cells from adult plp-sh ble-LacZ transgenic mice were injected into the telencephalon of P0Swiss or C3H/He mice. Detection of X-gal in brain sections of adult transgenic mice showed an intense staining of myelinated axonal tracts in the brain(bottom image). (C) Schematic representation of the localization of cell grafts in the analysed brain sections. Donor haematopoietic progenitors were inducedto differentiate into oligodendrocyte progenitors (blue dots), ependymocytes (red dots), neurons (green dots) and astrocytes (red asterisks).

576 S. Bonilla et al.

ã 2002 Federation of European Neuroscience Societies, European Journal of Neuroscience, 15, 575±582

Cells were kept cold during sorting, and sorted cells were recovered

in cold, complete RPMI 1640/FCS medium. Bone marrow sorted

cells (9 3 105) were resuspended in 6 mL of RPMI 1640 medium

and 2 mL (3 3 105 cells) were injected into the telencephalon of

cold-anaesthetized neonatal Swiss or C3H/He mice, using a Hamilton

syringe (Fig. 1B and C).

Intracerebral cell grafts

Neonatal (P0) Swiss mice were cold-anaesthetized and 2 mL of cell

suspension (3 3 105 cells) were introduced to the brain, through a

hole in the cranium, via a 10-mL Hamilton syringe under aseptic

conditions (Tables 1 and 2; Fig. 1B and C). The injection was

performed in the parietal area, 1 mm caudal and lateral to the skull

bregma point and 0.5 mm into the brain parenchyma from the dura

mater (Franklin & Paxinos, 1997). A control group of mice each

received 2 mL of RPMI 1640 medium without any cells, following

the same protocol (Table 1).

Four to six days after cell transplantation, experimental mice

anaesthetized with chloroform were ®xed by transcardiac perfusion

with 4% paraformaldehyde, in phosphate buffer (PB). The brains

were dissected out and post®xed overnight at 4 °C in the same

®xative. For vibratome sections, the ®xed brains were washed in

phosphate-buffered saline (PBS) and embedded in 4% agarose

(Sigma, Madrid) in PBS. Eighty-mm-thick sections were processed

for X-gal substrate detection as described previously (Spassky et al.,

1998). Brie¯y, brains were dissected in 0.1 M PBS, pH 7.4, ®xed by

immersion in 4% paraformaldehyde for 10 min at room temperature,

washed twice in PBS and stained for 6±15 h at 37 °C. The staining

solution contained (in mM): 5-bromo-4-chloro-3-indolyl-b-D-galacto-

side (X-gal) (United States Biochemical, Cleveland, OH), 2; potas-

sium ferrocyanide, 20; potassium ferricyanide, 20 and MgCl2, 2 in

PBS. Subsequently, the X-gal processed sections were immuno-

stained with O4 IgM monoclonal antibody (Chemicon, Temecula,

CA). The O4 mAb binds to oligodendroglial cells, including

progenitors and neuroepithelial precursors (Ono et al., 1997;

Spassky et al., 1998; Perez-Villegas et al., 1999).

In order to recognize all donor cells in experimental mice, we

performed an additional group of grafts (n = 12) using the C3H/He

mouse strain as the host (Fig. 1B and C). This mouse strain expresses

the H-2k haplotype in the H-2 histocompatibility complex, whereas

the plp-sh ble-LacZ mouse presents H-2b and H-2d haplotypes at the

same locus. Therefore, using a FITC-labelled 34-2-12 monoclonal

antibody (BD Pharmingen, San Diego, CA), which speci®cally

recognizes the alpha-3 domain of the H-2Dd antigen, we could detect

all donor cells, including those in which LacZ expression was not

driven due to the absence of PLP promoter signals. Three C3H/He

neonatal (P0) mice where injected with R2 cells from plp-sh ble-LacZ

mice. Control mice were injected as before with R1 cells (n = 4) or

2 mL of RPMI 1640 medium without any cells (n = 3). These

experimental mice were ®xed 5 days after graft and were processed

as cryotome sections for immunohistochemistry. First, the brains

were washed in PBS and cryoprotected overnight in 30% sucrose in

PBS. Then, serial sections were mounted in four parallel series and

processed for immuno¯uorescence with the 34-2-12 antibody. Double

immunolabelling was carried out with 34-2-12 antibody and the

following antibodies: O4, NG2 chondroitin sulphate proteoglycan

(anti-NG2 polyclonal antibody; Chemicon, Temecula, CA) which is a

marker for oligodendroglial and microglial cells (Reynolds & Hardy,

1997), antib-III-tubulin mAb (Eurogenec, Belgium), a speci®c

neuronal marker, and anti-glial ®brillary acid protein (GFAP) mAb

(Calbiochem, San Diego, CA), a speci®c marker for astrocytes.

Analysis of cell markers

The following murine monoclonal antibodies to speci®c antigens

were used: O4 (IgM isotype; (Chemicon, Temecula, CA), anti-H-

2Dd-FITC (IgG isotype; BD Pharmingen, San Diego, CA), antib-III-

tubulin antibody (IgG isotype; Eurogenec, Belgium). The anti-GFAP

is a rat monoclonal antibody (IgG isotype; Calbiochem, San Diego,

CA). The anti-NG2 is a rabbit polyclonal antibody. As respective

secondary antibodies we used biotinylated anti-mouse IgM, anti-

mouse IgG, anti-rat IgG and anti-rabbit IgG (Vector, Burlingame,

CA). The staining antibodies were streptavidin conjugated with

Peroxidase, Fluorescein or Texas Red (Vector, Burlingame, CA).

Parallel sections of all analysed cases were processed following an

identical protocol but without the step of incubation with ®rst

antibody and used as control for the speci®city of immunoreaction.

Results

Brain transplanted transgenic adult HSCs can be induced toexpress the LacZ-plp transgene

Bone marrow cells from the femur of plp-sh ble-LacZ mice (Spassky

et al., 1998), either in marrow tissue or in cell suspension, did not

react with X-gal in any of the controls previous to the transplant

experiments (n = 27; data not shown).

All cell grafts analysed were localized in the anterior pole of the

host striatum, under the anterior parietal cortex and at a distance of

0.5 mm from the ventricular lumen (Fig. 1C). Two different bone

marrow subpopulations (R1 and R2) were gated using a speci®c

sorting protocol and grafted into the brain of neonatal (P0) mice

(Fig. 1A and B). The R2 subpopulation was selected as an

experimental cell population, because it contained an enriched

population of c-kit+ (CD117+) cells (around 20%) in which both

primitive HSCs (CD117+, Sca-1+) and more differentiated haema-

TABLE 1. Presence of cell-type markers in analysed cases and information

about graft type and survival time after grafting

Casenumber

Survivalafter grafting(days)

Cell-type markers

X-gal O4 X-gal + O4 Ependymal cells

Experimental samples (R2 BM cells)PP1 4 ++ ND ND +PP2 4 ++ ++ + ±PP3 5 ++ ++ + +PP4 5 ++ ++ + +PP5 5 ++ ++ + ±PP6 6 ++ ND ND +PP7 6 ++ ++ + ±PP8 6 ++ ++ + ±PP9 6 ++ ++ + ±

Control samples (R1 BM cells)pp1 4 ± ND ND NDpp2, 4, 5 5 ± + ± ±pp3, 6 4±5 + ND ND NDpp7, 8, 9 6 ± + ± ±

Experimental cases are indicated with capital letters (PP) while control casesare indicated with small letters (ppn). Additional PBS controls carried out inparallel with R1 controls are not listed. All experimental grafts wereperformed at P0. BM, Bone marrow; ND, no data; +, Some positive cellsfor the analysed marker (»5% of the donor cells in the analysed region persection); ++, large cell number positive for the marker (> 60% of the donorcells in the analysed region per section); ±, labelled cells for the speci®cmarker are not detectable.

Haematopoietic progenitors differentiate into oligodendrocytes 577


topoietic progenitors (CD117+, Sca-1±) where present (Fig. 1A). The

R1 subpopulation was considered a control population as the CD117+

progenitor cells were poorly represented (less than a 3%). Three types

of grafts were performed (n = 9 for each type): R2 bone marrow

sorted cells, and two controls consisting of R1 sorted cells and 2 mL

of cell-free RPMI 1640 medium. Grafted mice were analysed at

different survival times (4±6 days post grafting). X-gal positive (X-

gal+) cells were observed in the brains (n = 9) grafted with the R2

population at 4±6 days post grafting (a mean of 60 6 10 cells at

4 days and 250 6 25 at 6 days; Table 1; Fig. 2A). In contrast, low

numbers of X-gal positive cells were detected in only two out of the

nine cases of R1 cell grafts (a total of 10 X-gal+ cells in each case).

X-gal reaction product was never observed following RPMI 1640

medium injections. Two populations of X-gal+ cells were observed in

relation with intracellular distribution of the X-gal precipitate; in the

centre of the injection area, spherical cells showed X-gal precipitate

in perinuclear and nuclear regions (a mean of 5% of X-gal positive

cells in R2 grafts and almost all the X-gal+ cells in R1 grafts). We

interpreted this population as remaining in¯ammatory donor cells in

the lesion area and they were not considered further in the present

work. The other X-gal positive population of cells, at the periphery of

the injection area, showed cytoplasm precipitates of X-gal reaction

product and were interpreted of oligodendroglial lineage. Six days

after grafting, these X-gal+ cells were observed to be undergoing

migratory movements, in which streams of X-gal+ cells followed the

axonal tracts of the corpus callosum and internal capsule (Fig. 2A). In

addition, in some cases (n = 5), we detected X-gal+ cells in the

ventricular epithelium, at some distance (400±800 mm) from the

injected area (Fig. 2G).

X-gal+ cells are also positive for O4

To con®rm the oligodendroglial nature of the X-gal+ cells, selected

sections of seven experimental brains presenting X-gal+ cells, and

sections of control brains (R1-injected, n = 8; RPMI 1640 medium-

injected, n = 6; Table 1) were processed for immunohistochemistry

using the O4 monoclonal antibody which binds to preoligodendro-

cytes and more mature oligodendrocytes. The O4 immunoreactivity

was detected in cells near the injection site, both in experimental

(Fig. 2B, C and F) and control (Fig. 2D and E) brains. X-gal+ cells in

experimental sections were either O4+ (» 40±50%; Fig. 2B, C and

F), or O4± (Fig. 2B, C and G). The presence of X-gal+/O4± cells may

be indicative of immature oligodendrocyte progenitors, which are still

not suf®ciently differentiated to bind O4 mAb, or alternatively, of

oligodendrocyte precursors in the neuroepithelium (Spassky et al.,

1998). In addition, we have the endogenous b-gal activity in immune

cells that were easily recognized by the perinuclear localization of X-

gal precipitate and their spherical morphology. In contrast, the X-

gal+/O4+ cells represent HSCs that had differentiated into cells of

oligodendroglial lineage. In R1- and RPMI 1640 medium-injected

control grafts, a number of host O4 immunoreactive cells appeared

exclusively in the periventricular zone close to the injection area

(Fig. 2D and E). These O4 immunoreactive cells were not observed

on the contralateral side, indicating that host oligodendrocyte

progenitors were locally stimulated to initiate their maturation in

response to the injection procedure itself. In only two out of nine R1

control cases, O4 positive cells (eight cells) were observed to

coexpress X-gal (X-gal+/O4+).

Adult HSCs can be induced to differentiate intooligodendrocytes, ependymocytes, astrocytes and neurons

To explore the possibility of generation of other neural phenotypes

from HSCs, in addition to oligodendrocyte progenitors, we used the

C3H/He mouse strain as hosts for plp-sh ble-LacZ transgenic HSCs.

Experimental and control grafts were analysed after 5 days of

survival. In the ®ve R2 grafted cases, some 34-2-12+ cells were found

to be integrated into the ventricular epithelium of the lateral ventricle

showing a columnar morphology, characteristic of ependymal cells

(Fig. 3A±D). From these ventricular integrated cells, subventricular

and centrifugal migrating cells can be observed to invade more

super®cial neural domains and probably differentiate into other cell

types (Fig. 3A±D). Other donor cells were detected in: anterior

telencephalic subventricular zones; in the cortex; the striatum and in

the main axonal tracts (Fig. 1C). In sections containing 34-2-12+

cells, double immunostaining was performed with O4, NG2, anti-b-

III-tubulin (b-Tub) and anti-GFAP antibodies. Con®rming our

previous results, we detected, by double-label immunohistochemistry,

cells which were immunoreactive with both 34-2-12 and O4

antibodies (Fig. 3A±P). These cells are representative of oligoden-

FIG. 3. Hematopoietic stem cells can be induced to develop as ependymocytes and to express oligodendrocyte markers. (A±D) Ependymal cells. (A) Confocalmicroscopic analysis of donor cells (green ¯uorescence) integrated into the ventricular epithelium of a C3H/He mouse. Double immunostaining with the O4oligodendroglial-speci®c antibody shows that some ventricular and subventricular cells are from the graft and have developed an oligodendroglial phenotype(yellow ¯uorescence, arrows), while other donor cells in this region are O4± (arrowhead). Dotted line indicates the line of ventricular lumen; (B±D) Sectionsof grafted brains showing the ependymal integration of grafted cells (arrows in B and green cells in D). (E±N) Oligodendrocyte progenitors. (E and F) A 1-mm section analysed by confocal microscopy, showing a donor cell (arrow) with double labelling in F (arrow); the arrowhead indicates a hosts O4+ cell. (G±I) Section analysis (1 mm thick) illustrating the donor cell channel (G, green), the O4 channel (E, H, red) and double stained oligodendrocyte progenitors (I,yellow). (J and K) Some grafted cells in host anterior commissure (J) were NG2+ (K). (L±N) Sections showing donor cells (L, green) and O4+oligodendrocytes (M) in the periphery of the injection area, several oligodendrocytes were differentiated from donor cells (N, arrows showing yellow cells).(O±R) Pictures from the injection area and the lateral ventricle (lv), (O). (Q) The distribution of donor cells (green ¯uorescence). (P) The analysis ofoligodendrocyte distribution (O4+ cells) in the same section shown in O. (R) The absence of non-speci®c cross-reactivity of neural cells when the sectionswere not incubated with O4 antibody. Few spherical cells are stained in the centre of the injection suggesting its in¯ammatory character. Scale bar, 10 mm,(A and G±I); 100 mm, (B±D, J and K and O±R); 40 mm (E, F and L±N).

FIG. 2. b-gal activity in cells of oligodendroglial lineage generated from HSCs (A±C). X-gal+ oligodendroglial progenitors were detected both in the injectionarea (arrow) and migrating through the internal capsule (ic; arrowheads) in a representative section of case PP7. (B) Represents a high power magni®cation ofthe ®eld shown in C (see large arrowhead). Arrows indicate X-gal+ (blue/green)/O4+ (brown) oligodendrocyte progenitors. (D and E) Section from braininjected with control R1 cells (control case pp3). No X-gal+ cells were detected, but O4 immunoreactivity was observed to be locally increased in thesubventricular zone close to the injection area. (E) Enlargement (320) of the insert shown in D. (F) Oligodendrocyte progenitors (X-gal+) from the donormarrow cells coexpress O4 (arrows point to the cell nucleus and arrowheads indicate the double labelled cytoplasm). The empty arrow shows a spherical X-gal+/O4± cell of possible immune character. (G) An X-gal+/O4± cell in the ventricular epithelium. Arrows indicate the thickness of the ventricularepithelium. cc, corpus callosum; ic, internal capsule; lv, lateral ventricle; ST, striatum; SVZ, subventricular zone; VE, ventricular epithelium. Scale bars,25 mm (A±D); 20 mm (F and G).



Fig. 3



droglial differentiation from HSCs. The local oligodendroglial

reaction in the host and donor cells, induced to differentiate into

oligodendrocytes (Fig. 3O and P), was not detectable when parallel

experimental sections (Fig. 3Q) were not incubated with the antibody

(Fig. 3R). The b-III-tubulin immunoreactivity was also observed in

some donor cells (Fig. 4A±F). The analysis of GFAP immunoloca-

lization revealed that some 34-2-12+ cells, were GFAP positive as

well. In addition, the area near to the injection side showed an intense

astroglial reaction (Fig. 4O), where some astrocytes were from the

donor. Grafted cells detected in the ependymal layer showed two

types of phenotype (Fig. 3A and B): an O4+ population, which can be

characterized as a population of O4+ oligodendrocyte precursors

(Ono et al., 1997; Spassky et al., 1998), and O4± ependymocytes,

indicative of a more undifferentiated population. Donor cells with an

immature aspect, localized in the subventricular zone and dispersed

in the striatum (Fig. 4A±D) and in the cerebral cortex (Fig. 4E and

F), were found to express b-III-tubulin, indicative of their neuronal

character. In four experimental cases, NG2 inmunoreaction was

observed in several groups of 34-2-12 positive cells (Table 2). In

axonal tracts, like anterior commissure and internal capsule, scattered

cells express the two markers that in addition to their multipolar

morphology and localization suggest an oligodendroglial character

(Fig. 3J and K), whereas small NG2/34-2-12 positive cells are

dispersed around the injection area and the local blood vessels,

showing an interstitial and perivascular microglial character (data not

shown). In the corresponding controls, some NG2/34-2-12 positive

cells were observed exclusively around the injection area, but all of

then showed small size suggesting a microglial character (data not

shown).

R1-grafted mice showed clusters of spherical 34-2-12 immunopo-

sitive cells in the injection area, but very few were observed outside

the injected cell clusters. These cells did not express b-III-tubulin,

and few of them were O4+ (a mean of six cells were found in four

consecutive sections) or GFAP+ (a mean of ten cells in three

FIG. 4. Hematopoietic stem cells can be induced to develop as neurons and astrocytes. (A±F) Neurons. (A±D) In the host striatum several donor cells(indicated by arrows in A±D) double stain with the neuronal marker, b-III-Tubulin (yellow cells labelled by arrows). (E and F) Confocal analysis of anexperimental brain section at the level of the parietal cortex (super®cial to the grafted domain). A grafted cell in the super®cial areas of the cortical plate (E)showed b-III-tubulin immunoreactivity (F) and migratory morphology. The arrowhead indicates a grafted b-III-tubulin negative cell. (G±S) Astroglial cells.(G±I and L±N) 1-mm-thick confocal section series showing donor cells (green channel) and astroglial cells (red channel); donor cells that express GFAPappeared in yellow in I and N. (J and K) High magni®cation of a donor cell expressing the astroglial marker. (O and P) Low power pictures showing theintense astroglial reaction in the host injection area; the yellow dots indicate that some donor cells have been transformed into astrocytes. (Q) Host (red) anddonor (yellow) vascular feet are observed around host brain blood vessels (BV). (R and S) Some donor cells (green) in the astroglial reaction zone (red) donot express GFAP. Scale bar, 20 mm (A, B, E, F, G±I, L±N, R and S). 40 mm, (C and D); 10 mm, (J and K); 100 mm, (O, P).



consecutive sections). We did not observe R1 donor cells integrated

into the ventricular epithelium or in the subventricular zone (data not

shown).

Discussion

Our data strongly indicate that a subpopulation of adult mouse HSCs

retains the capacity to differentiate into cell types other than blood

cells, and more speci®cally into oligodendrocyte progenitors,

ependymal cells, neurons and astrocytes. Thus, in adults, intrinsic

genetic mechanisms of cell lineage restriction and cell fate commit-

ment seem to retain some plasticity, in vivo. Although we have not

fully identi®ed the bone marrow subpopulations that differentiate into

oligodendrocytes and ependymocytes, it is quite reasonable to

suppose that the c-kit+ (CD117+) cells which are enriched in the

R2 sorted population with respect to the R1 population, were those

which differentiated into CNS cells. In support of this interpretation,

very few oligodendrocytes differentiated from the grafted R1 cells, in

which CD117+ cells represent a minor percentage. The clonogenic

HSC has been reported to be included in the CD117+/Sca-1+

subpopulation that can be separated as long-term HSC, short-term

HSC and multipotent progenitors (MPP), which do not have self-

renewal potential (Lagasse et al. 2001). As R1 and R2 subpopulation

isolation were based on their size, both CD117+/Sca-1+ and CD117+/

Sca-1± (committed haematopoietic population) cells present in R2 are

not only quantitatively higher than in R1 but also could be

qualitatively different; being more plastic and able to differentiate

into oligodendrocytes. Alternatively, other cells included in the R2

subpopulation (CD117± cells) could have the potential to differen-

tiate into oligodendrocytes. However, we do not favour this

hypothesis as the ability to transdifferentiate in other systems

(generation of mature hepatocytes in the liver of tyrosinemic mice)

has been reported to reside in the CD117+ population upon adult

bone marrow injection (Lagasse et al. 2000).

To our knowledge, this is the ®rst report of in vivo differentiation

of HSCs into oligodendroglial progenitors, which are able to

proliferate and migrate like normal oligodendrocyte progenitors.

Previous works have only reported the differentiation of MSCs into

astrocytes, neurons and microglia, but oligodendrocytes were not

clearly identi®ed (Eglitis & Mezey, 1997; Azizi et al., 1998; Kopen et

al., 1999; Brazelton et al. 2000; Mezey et al. 2000). Mezey &

Chandross (2000) suggest the possibility that BMCs differentiate into

oligodendrocytes in the case where some donor cells migrate into

host axonal tracts, but they do not speci®cally characterize them.

Azizi et al. (1998), Kopen et al. (1999) and Mezey et al. (2000) did

not report the emergence of oligodendrocytes from HSC in bone

marrow (Mezey et al. 2000) or stromal (Azizi et al., 1998; Kopen et

al., 1999) transplants. Nevertheless, Eglitis & Mezey (1997),

Brazelton et al. (2000) and Mezey & Chandross (2000) grafting

unselected BMCs, where some population of HSCs should be present,

could not clearly detect oligodendrocytes either. Therefore, we can

conclude that our observation of HSCs transformation into oligoden-

drocytes is due to our more sensitive methodology and/or our active

searching for oligodendrocyte lineage markers in donor cells. Our

new experimental model employing the plp-sh ble-LacZ transgenic

mouse, that provides an internal marker for cells of the oligoden-

drocyte lineage, together with the use of the C3H/He mouse as host,

has allowed us to obtain more molecular and morphological

resolution of cell fate analysis than previous studies.

Sasaki et al. (2001) have recently reported the existence of

oligodendroglial potential in spinal cord BMCs grafts after

demyelinating lesions. Conversely, they did not detect oligodendro-

cytic cells when selected haematopoietic progenitors where injected

in their experimental model. The differences between this result and

our data can be explained by the existence of different environmental

factors that control HSC differentiation in the adult spinal cord and

the neonatal brain (non-equivalent pathways) or by the heterospeci®c

character of their model.

The detection of some neuronal features in donor cells, after

intravascular injection of selected stromal or unselected bone marrow

cells in PU.1 mutants or lethally irradiated mice, has recently been

reported (Brazelton et al. 2000; Mezey et al. 2000). The possibility of

neuronal generation in the subventricular zone and active centrifugal

migration from ependymal integrated cells were also suggested by

Mezey et al. (2000). The pluripotent and stem cell character of

ependymal cells has been reported recently by Rietze et al. (2001).

Our results are indicative of the recapitulation of some embryonic

stages of normal cell differentiation and migration in the subven-

tricular layer. We have studied brains at a very early stage post-

grafting and, therefore, we were able to detect the initial events of

stem cell integration into the host brain and primary neuronal

production. However, further studies using our experimental system,

with long-term survival animals, will be important to reveal the

possible functional integration of neuronal marrow derivatives in the

host brain.

Bone marrow stem cells can be useful in the treatment of a wide

variety of neurological diseases, offering signi®cant advantages over

TABLE 2. Presence of cell-type markers in cells from C3H/He mice and information about graft type and survival time after grafting

Casenumber

Survivalaftergrafting(days)

Cell-type markers

34-2-12 O4 b-Tub NG-234-2-12/NG-2

34-2-12/O4

34-2-12/b-Tub

Ependymalcells

Experimental samples (C3H/He mouse strain)SB39 5 ++ ++ ++ ND ND ++ ++ ++SB41 5 ++ ++ ++ ++ ++ ++ ++ ++SB43 5 ++ ++ ++ ++ ++ ++ ++ ++SB53, 56 5 ++ ++ ND ++ ++ ++ ND ++

Control samples (R1 BM cells)sb38, 44, 45, 62 5 ++ + ± + + + ± ±

Experimental cases are indicated with capital letters (SB) while control cases are indicated with small letters (sb). Additional PBS controls carried out in parallelwith R1 controls are not listed. All experimental grafts were performed at P0. BM, Bone marrow; b-Tub, b-III-tubulin; ND, not tested; +, Some positive cells forthe analysed marker (»5% of the donor cells in the analysed region per section); ++, large cell number positive for the marker (> 60% of the donor cells in theanalysed region per section); ±, labelled cells for the speci®c marker are not detectable.



other stem cell types. Indeed, HSCs are readily accessible and

overcome the risks associated with obtaining neural stem cells, and

the ethical concerns associated with the use of fetal tissue. In

comparison to MSC, HSCs may be more accessible for repetitive

therapeutic trials as they can be easily obtained from the peripheral

blood and do not require bone marrow extractions. The bene®ts of

bone marrow autotransplants in acute phases of the disease process

have been reported in MS. The advantage of this protocol derives

from the absence of myelin speci®c T cell clones in the bone marrow

graft (reviewed in Burns & Burt, 1999; Saiz et al. 2001). Regarding

the data on the potential of MSCs and the possibility of crossing the

blood±brain-barrier (Brazelton et al. 2000; Mezey et al. 2000), it is

possible that grafted HSCs could preferentially migrate to the active

plaques in MS. Then, differentiate into oligodendrocyte progenitors,

which in turn, could remyelinate and induce trophic effects to

affected axons. At present, we are currently exploring the possibility

of remyelinating axons in disseminated in¯ammatory plaques by

intravascular perfusion of heterologous HSCs (P. AlarcoÂn, R.

Villaverde and S. MartõÂnez, unpublished observation). It will also

be of interest to explore the therapeutic possibilities associated with

increasing host stem cells in peripheral blood in animal models. This

could be a promising cell therapy for MS as well, as autogenic grafts

of normal or manipulated stem cells could regenerate disseminated

lesions.

Acknowledgements

The authors wish to express their thanks to C. Sotelo for his critical andconstructive comments, to B. Zalc (INSERM U-495 HoÃpital de la SalpeÂtrieÁre,Paris) for plp-sh ble-LacZ transgenic mice, to Pedro Lastres for cytometryanalysis and sorting and to Monica RoÂdenas for her technical contribution andexpert assistance. This work was ®nanced by the following grants: DIGESIC-MEC PM 98±0056 and PM98-0052 from the Spanish Ministry of Educationand Culture; 708/CV/99 from the Seneca Foundation; 1FD97-2090 and 1FD-2061 from FEDER and BIO4-98±0309, QLG2-CT-1999±00793, QLRT-1999±31556 grants from the European Community. The work carried out by A.S.was ®nanced by grant SAF2000.118.CO3.3. Finally we would like to thank theagency ACTS ([email protected]) for revising the ®nal version of this paper.

Abbreviations

Ac, anterior commisure, b-Tub, b-III-tubulin; BMC, bone marrow cell; BV,blood vessel; CC, corpus callosum; Chi, hippocampal commissure; Cx, cortex;DT, dorsal thalamus; ET, epithalamus; Fx, formix; GFAP, glial ®brillary acidprotein; HSC, bone marrow hematopoietic stem cell; ic, internal capsule; lv,lateral ventricucle; mAb, Monoclonal antibody; ml, midline; MS, MultipleSclerosis; MSC, bone marrow stromal stem cell; PBS, phosphate-bufferedsaline; pi, pial surface; S, septum; ST, striatum; SVZ, subventricular zone; VE,ventricular epithelium.

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haematopoietic progenitor cells from adult bone marrow differentiate into cells that express...

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