Review

Neural Cells Derived From Adult BoneMarrow and Umbilical Cord Blood

Juan R. Sanchez-Ramos*Center of Aging and Brain Repair, University of South Florida and James Haley VA Hospital Health ScienceCenter, Tampa, Florida

Under experimental conditions, tissue-specific stemcells have been shown to give rise to cell lineages notnormally found in the organ or tissue of residence. Neuralstem cells from fetal brain have been shown to give riseto blood cell lines and conversely, bone marrow stromalcells have been reported to generate skeletal and cardiacmuscle, oval hepatocytes, as well as glia and neuron-likecells. This article reviews studies in which cells frompostnatal bone marrow or umbilical cord blood wereinduced to proliferate and differentiate into glia and neu-rons, cellular lineages that are not their normal destiny.The review encompasses in vitro and in vivo studies withfocus on experimental variables, such as the source andcharacterization of cells, cell-tracking methods, andmarkers of neural differentiation. The existence of stem/progenitor cells with previously unappreciated prolifera-tion and differentiation potential in postnatal bone mar-row and in umbilical cord blood opens up the possibilityof using stem cells found in these tissues to treat degen-erative, post-traumatic and hereditary diseases of thecentral nervous system. © 2002 Wiley-Liss, Inc.

Key words: bone marrow stromal cells; neuronal differ-entiation; stem cells

Two conceptual pillars of developmental biologyhave been rocked in the last few years. The long standingdogma that neurons in the adult brain do not regeneratehas been challenged by evidence that new neurons areborn in “germinal zones” of the hippocampus and sub-ventricular zone in rodents and humans throughout life(Eriksson et al., 1998; Gage, 2000). The second conceptunder attack is that a cell committed to a specific pheno-typic fate by virtue of residence in a mature organ cannotchange its destiny. A growing body of experimental evi-dence proves that cells derived from one adult tissue canchange into cellular phenotypes not normally found in thattissue. This suggests that cells from mature tissues can bereprogrammed (“transdifferentiated”) to change into acompletely distinct phenotype from that found normallyin that tissue. An alternative explanation is that maturetissues harbor a small number of pluripotent stem cells

with a greater differentiation potential than appreciatedpreviously.

A stem cell is defined by its functional capacity toboth self-renew and to generate a large number of differ-entiated progeny (McKay, 1997; Gordon and Blackett,1998; Scheffler et al., 1999). The truly totipotent cell,capable of generating all cell types and constructing acomplete organism, is the fertilized egg. During develop-ment and morphogenesis cells proliferate, migrate anddifferentiate, but throughout this process there is a residualof quiescent, uncommitted cells that are believed to be theresult of asymmetric division ensuring self-renewal of thestem cell population. Stem cells can be isolated from thedeveloping embryo and from specific adult tissues, buttheir potential for differentiation into all cell types be-comes restricted gradually to a more limited range of cellstypical of the mature tissue in which the stem cell (orprogenitor cell) resides.

Over the last few years, tissue-specific stem cells havebeen shown to give rise to cells not normally found in theorgan or tissue of residence. For example, neural stem cellscan give rise to blood cell lines (Bjornson et al., 1999) andbone marrow stromal cells can generate skeletal muscle(Wakitani et al., 1995; Ferrari et al., 1998), cardiac muscle(Orlic et al., 2001a,b; Makino et al., 1999), oval hepato-cytes (Petersen et al., 1999), as well as glia and neuron-likecells. The existence in postnatal tissue of stem/progenitorcells with ample proliferation and differentiation potentialopens up the possibility of using autologous adult stem

Contract grant sponsor: Helen E. Ellis Research Endowment; Contractgrant sponsor: VA Merit Review; Contract grant sponsor: NIH/STTR;Contract grant sponsor: Layton BioSciences, Inc.; Contract grant sponsor:Saneron-CCell Biotherapeutics, Inc.

*Correspondence to: Juan R. Sanchez-Ramos, Ellis Professor of Neurol-ogy, Director of Stem Cell Research, Center of Aging and Brain Repair,University of South Florida, and James Haley VA Hospital Health ScienceCenter MDC 55, 12901 Bruce B. Downs Blvd., Tampa, FL 33612.E-mail: jsramos@hsc.usf.edu

Received 6 March 2002; Revised 15 April 2002; Accepted 16 April 2002

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jnr.10337

Journal of Neuroscience Research 69:880–893 (2002)

© 2002 Wiley-Liss, Inc.

cells to treat degenerative, post-traumatic and hereditarydiseases. This article reviews studies in which cells frompostnatal bone marrow or umbilical cord blood wereinduced to proliferate and differentiate into glia and neu-rons, cellular lineages that are not in their normal reper-toire.

MULTIPOTENT NON-HEMATOPOIETICPROGENITORS IN BONE MARROWBone marrow stromal cells (BMSC) provide the

structural and functional support for the generation ofblood cell lineages from hematopoietic stem cells.BMSC consist of morphologically and biochemicallydistinct cell types: bone marrow fibroblast–reticularcells, adipocytes, osteoblasts, macrophages, and endothelialcells. BMSC can be cultivated in vitro and contain pro-genitors capable of generating bone, cartilage, fat, andother connective tissues. These non-hematopoietic pre-cursors found in bone marrow stromal cells are also knownas colony forming unit fibroblasts (CFU-f) and mesen-chymal stem cells (MSC). (Prockop, 1997). The BMSCper se have been reported to have many characteristicsof MSC; hence, the terms are often used synonymously.MSC, a homogenous population of fibroblast-like cellspurified by Percoll gradient and expanded in vitro cangenerate progeny that differentiate into multiple celllineages, including bone, fat, tendon and cartilage (Pit-tenger et al., 1999). Several recent reports demonstrate

that under specific experimental conditions BMSC canalso differentiate into cells that are not part of theirnormal repertoire: skeletal and cardiac muscle, hepato-cytes, glia and neurons (Azizi et al., 1998; Ferrari et al.,1998; Makino et al., 1999; Petersen et al., 1999;Sanchez-Ramos et al., 2000; Jackson et al., 2001; Ko-hyama et al., 2001; Orlic et al., 2001a,b).

Reports on neural cell differentiation from bonemarrow or umbilical cord blood can be grouped foranalysis: the in vitro studies (Table I) and the transplan-tation studies (Table II). To facilitate comparison ofexperimental results from different groups, Table I listssalient experimental variables used in each study. Mostresearchers relied on the separation of stromal cells byadherence to plastic, and in most of the in vitro studieshematopoeitic progenitors were depleted or removedfrom the marrow cells. Nevertheless, one study identi-fied the presence of CD34� cells in the subclone of cellsthat could give rise to neural cells. The agents used toinduce neural differentiation in vitro included retinoicacid, growth factors (alone or in combination), antioxi-dants, a demethylating agent, compounds which in-crease intracellular cyclic AMP (cAMP) and a physio-logical neural inducer, noggin. Although both neuron-like cells and glia were generated in most of the in vitrostudies reviewed, glial cells were generated exclusivelyin one study (Nakano et al., 2001). Neurons with no

TABLE I. Differentiation of Bone Marrow Cells to Neural Lineages In Vitro: Comparison of Study Parameters

Population of cells Proliferation media Induction of differentiation Markers of neural lineages Reference

Human BMSC depleted ofCD34� and mouse BMSCdepleted of Scal� cells; plasticadherent cells

DMEM � FBS �EGF � FGF

RA � BDNF � NGF; onpolyethylimine-coatedsubstrate or bed ofmurine fetal midbraincells

Nestin, �-tubulin III,NeuN, GFAP

Sanchez-Ramos et al., 1998,2000

Rat BMSC depleted of CD11band CD45; clones of BMSCproduced by limiting dilution;plastic adherent human BMSC

�-MEM �DMEM � FBS

�-mercaptoethanol,DMSO/BHA

Nestin, NSE, NF-M,TrkA (no GFAP� cells)

Woodbury et al., 2000;Black and Woodbury,2001

Mouse BMSC clones produced bylimiting dilution; each clonecharacterized with surfacemarkers; 2 of 3 clones wereCD34�, Scal� CD140�,CD44�, and CD140�

IMDM � FBS 5-Aza-C (4 days) thenNGF � BDNF �NT3; noggin onfibronectin-coatedsubstrate

TuJ-1, NeuN, Hu,GFAP, Gal-C, trkA,trkB, trkC, NCAM,GAP-43

Kohyama et al., 2001

Human BMSC depleted ofCD45� glycophorinA� cells;passaged for 20–70 doublings;plastic adherent

Low glucoseDMEM � FBS� EGF �PDGF; passagedwhen 50%confluent

bFGF 3 weeks;fibronectin-coatedsubstrate

�-tubulin III, NSE,glutamate, GFAP,Gal-C, MAP

Reyes and Verfaillie, 2001

Human BMSC �-MEM � FBS;passaged when70–90%confluent

Isobutylmethylxanthine/Dibutyryl cAMP

NSE, vimentin Deng et al., 2001

Human umbilical cord bloodmononuclear cells; plasticadherent

DMEM � FBS �EGF � FGF

RA � NGF Musashi1, Nestin, �-tubulin III, NeuN,GFAP

Sanchez-Ramos et al., 2001

Neural Cells Derived From Bone Marrow 881

glia were uniquely produced in another study (Wood-bury et al., 2000). One in vitro study provided physi-ological evidence of neuronal function after differenti-ation from BMSC. Many experiments were performedin vivo and involved transplantation of untreated ormodified marrow cells into a host animal. Table IIsummarizes the experimental variables in most of thetransplantation studies. Bone marrow was obtained

from mice, rats or humans. In mice, some researcherspretreated animals with 5-fluorouracil to increase thegeneration of marrow stem cells. Cells were implanteddirectly into the brain or ventricles, or administeredsystemically via a tail vein. Various animal models wereused: rats with ischemic infarct, irradiated mice,MPTP-induced parkinsonism and immunocompro-mised mice. Improvement in neurologic deficits were

TABLE II. Differentiation of Bone Marrow Cells to Neural Lineages After Transplantation

Population of cells/ sourceMarker of donor

marrow cellsAnimal model/route of

deliveryMarkers of neural

lineages Reference

Bone marrow mononuclearcells from male micepretreated with 5-fluorouracil

Cells labeled withretroviralvector carryingneomycinR

gene; Y-chromosome

Sublethally irradiated femalemice; cells administeredvia tail vein

GFAP, F4/80 antigen(microglial marker)

Eglitis and Mezey,1997

Human BMSC separatedby adherence to plastic

Bisbenzamide Normal rat; cells grafted intostriatum

Migration of BMSC“similar to astrocytes”

Azizi et al., 1998

Bone marrow mononuclearcells from male ratspretreated with 5-fluorouracil

Y-chromosome Stroke in spontaneouslyhypertensive female rats;irradiated; infused tail vein

GFAP Eglitis et al., 1999

BMSC from FVB/N mice BrdU orbisbenzamide

Neonatal mouse; cellsinfused into lateralventricle

GFAP, Nf Kopen et al., 1999

Unprocessed bone marrowcell suspensionscontaining 107 cells/animal from wild-typemale mice

Y-chromosome Female mouse PU.1knockout strain lacksmacrophages, neutrophils,mast cells, osteoclasts, B-and T-cells at birth; cellsgiven intraperitoneally

NeuN, NSE Mezey et al., 2000

Bone marrow mononuclearcells from GFPtransgenic mice

GFP� cells Lethally irradiated isogenicmice; cells administeredvia tail vein

NeuN, Nf-H, �-tubulinIII; no GFAP� cellscolabeled with GFPfound

Brazelton et al., 2000

Bone marrow mononuclearcells from C57/bl micepretreated with 5-fluorouracil andprestimulated with IL-6and murine stem cellfactor on fibronectincoated substrate

Cells labeled withretroviralvectorcontainingGFP

Sublethally irradiated mice;cells infused via tail veinor grafted into striatum

GFAP, CAII, Iba1colabeled with GFPafter direct grafting; noneuronal markersfound

Nakano et al., 2001

BMSC male rat pretreatedwith 5-fluorouracil

Y-chromosome Head trauma model femalerats; cells infused via tailvein

NeuN, GFAP Mahmood, 2001

Human umbilical cordmononuclear

Anti-humannucleimonoclonal Ab

Stroke model male rats; cellsinfused via tail vein

NeuN, MAP-2, GFAP Chen et al., 2001b

BMSC; mouse pretreatedwith 5-fluorouracil

BMSC labeledwith BrdU

MPTP mouse model ofParkinson disease; cellsgrafted in striatum

Tyrosine hydroxylase Li et al., 2001

BMSC; rat pretreated with5-fluorouracil

BMSC labeledwith BrdU

Stroke model male rats; cellsinfused in carotid

MAP-2, GFAP Li et al., 2000

Human umbilical cordblood stromal cellscultured on plastic inDMEM or RA � NGF

Anti-humannucleimonoclonal Ab

1-day-old neonatal rat pups;intraventricular injection

TuJ 1, GFAP Zigova et al., 2002

882 Sanchez-Ramos

demonstrated in the stroke, trauma and Parkinson dis-ease animal models.

DIFFERENTIATION OF BONE MARROWCELLS INTO MICROGLIA, ASTROCYTES

AND OLIGODENDROGLIAThe earliest work on the relationship between mar-

row cells and glial cells focused on the natural trafficking ofmicroglia between bone marrow and brain. Microglia arebelieved by many to derive from an hematopoietic line(monocytes), whereas astrocytes and oligodendroglia areconsidered to be derived from embryonic neuroectodermand are developmentally distinct from microglia (Ling andWong, 1993; Ling, 1994). Some microglia, however, arethought to have a neuroectodermal origin (Neuhaus andFedoroff, 1994). Eglitis and Mezey (1997) sought to de-termine the extent to which cells outside the centralnervous system contribute to the maintenance of microgliain adult mice. Bone marrow cells were labeled with aretroviral vector carrying the gene for neomycin resistance(neoR). A second approach for tracking marrow-derivedcells in the recipient relied on in situ hybridization with aprobe specific to the Y-chromosome of male donor mar-row cells; the neoR- labeled male bone marrow cells werethen infused by tail vein into sublethally irradiated femalemice (WBB6F1/J-KitW/KitW-v). Over the next few daysto weeks later, there was an influx of labeled cells into thebrain of the recipients (Eglitis and Mezey, 1997). Marrow-derived cells were found throughout all regions of thebrain from cortex to brainstem. They appeared to residewithin the parenchyma because perfusion with phosphate-buffered saline (PBS) did not remove them. Occasionalmarrow-derived cells were found in association with vas-cular structures. The densities of donor cells in the recip-ient brain parenchyma paralleled the vascularity of a givenregion. Cortex, with few capillaries, had a lower celldensity of marrow-derived cells than the more vascular-ized chorid plexus. Area postrema had the highest densityof marrow-derived cells within the parenchyma. Somebone marrow-derived cells were positive for the micro-glial antigenic marker F4/80. Other marrow-derived cellsexpressed the astroglial marker glial fibrillary acidic protein(GFAP). Approximately 10% of the marrow-derived cellsin the brain expressed either the microglial F4/80 antigenor GFAP; the identity of the remaining 90% of themarrow-derived cells in the recipient brain is unknown.These results indicated that some microglia and astrogliaarose from a precursor that is a normal constituent of adultbone marrow. The authors considered the appearance ofmarrow-derived astroglia a normal process because thenumbers of marrow-derived cells detected in brain in-creased over time, and their appearance did not appear tobe a consequence of the transplantation procedure. Inaddition, the radiation did not appear necessary for themarrow cells to migrate to brain. Male donor cells en-grafted and persisted for greater than two months in re-cipients that had received no irradiation. Furthermore,there were as many Y-chromosome/GFAP double-stained cells seen in the animals without radiation as seen

in animals with irradiation. An interesting observationmade by the authors was the appearance of cells withmarrow markers in the ependymal layer of the ventricles.The finding of bone marrow-derived cells suggest thatthese cells home in and differentiate in response to signalsfrom the subependymal zone.

In another set of experiments using the male bonemarrow to female recipient paradigm, Eglitis et al. (1999)demonstrated a preferential homing of marrow-derivedprogenitors to the site of an hypoxic/ischemic injury in ratbrain. In an acute unilateral middle cerebral artery occlu-sion model, 2.8% of the total DAPI-stained nuclei countedin the ischemic lesioned side of the brain were derivedfrom bone marrow (i.e., Y-chromosome�), whereas 1.8%of the total nuclei were bone-marrow derived in the intactunlesioned side. Thus, the ischemic side of the brain hadattracted 55% more marrow-derived cells than the non-ischemic side. No such difference was found between thetwo hemispheres in brain sections obtained from twointact animals grafted with bone marrow cells. The per-centage of total GFAP� cells that were double labeled(Y-chromosome�/GFAP�) was 161% greater in the le-sioned hemisphere compared to the unlesioned side. Ofthe total GFAP� cells on the lesioned side, 4.7% werebone marrow-derived, and on the unlesioned side, 1.8%were bone marrow-derived.. There was clearly a prefer-ential targeting of the marrow-derived astrocytes to aregion of cerebral injury. Astrocytic proliferation has beenshown to occur in both the ischemic regions of brain aswell as in regions that are undamaged by ischemia. Thefindings of Eglitis et al. (1999) suggest that an additionalsource of astrocytes is related to increased migration anddifferentiation of cells derived from bone marrow.

Other researchers have reported that infusion of hu-man BMSC into rodent brain resulted in engrafting, mi-gration and survival of cells (Azizi et al., 1998; Kopen etal., 1999). A subset of human marrow cells (separated onthe basis of adherence to plastic) labeled with bisbenz-amide were injected directly into the corpus striatum ofrat. From 5–72 days later, brain sections were examinedfor the presence of donor cells; approximately 20% of theinfused cells had engrafted in the host brain. The cells hadmigrated from the injection site to corpus callosum, con-tralateral cerebral cortex and ipsilateral temporal lobe. Af-ter engraftment, these cells lost markers typical of marrowstromal cells in culture, such as immunoreactivity to an-tibodies against collagen and fibronectin. BMSC devel-oped many of the characteristics of astrocytes, and theirengraftment and migration contrasted markedly with fi-broblasts that continue to produce collagen after implan-tation.

Grafting of a subset of BMSC into the lateral ven-tricle of neonatal mice also resulted in their migrationthroughout the forebrain and cerebellum without disrup-tion of host brain architecture (Kopen et al., 1999). Inthese experiments, the bone marrow stromal cells weredepleted of cells that express the cell surface receptorCD11b, a marker of myelopoietic cells. The grafted

Neural Cells Derived From Bone Marrow 883

BMSC were labeled with bisbenzamide or bromode-oxyuridine (BrdU) to track the fate of the cells. In theforebrain, a large number of donor cells were found ipsi-lateral to the injection site throughout the striatum, fromthe anterior commissure to the cingulate cortex. BMSCwere also reported to line white matter tracts, includingthe corpus callosum and the external capsule, suggestingthat their distribution throughout the forebrain was anordered process of migration. Many BMSC were detectedlining the ependyma throughout the ventricles. The pres-ence of cells double-labeled for BrdU and GFAP sug-gested that some of the BMSC within the corpus striatum,the molecular layer of the hippocampus and the cerebel-lum had differentiated into astrocytes. Interestingly,BMSC were also located in areas undergoing active post-natal neurogenesis, including the Islands of Calleja in theventral forebrain and the subependyma of the olfactorybulb. A large number of BrdU-labeled cells were foundintegrated within the folia of the cerebellum. The majorityof the cells were localized to the external granular layer,the internal granular layer and to a lesser extent, themolecular layer. The Purkinje cells in the cerebellum werenot labeled with BrdU, consistent with their earlier mat-uration during embryogenesis. Most of the BrdU-labeledcells in the cerebellum were found ipsilateral to the side ofinjection, but some were found on the contralateral side.Many BrdU-labeled BMSC lined the fourth ventricleuniformly, and small foci were seen in the white mattertracts adjacent to the dorsal horns of the fourth ventricle.The authors suggested that the BMSC gained access to theexternal granular layer and the reticular formation of thebrain stem by following a pathway similar to that used byneural progenitors at the time when they emigrate out ofthe rhombic lip, into the primordial external granular layerduring embryogenesis. In rare sections, occasional neuro-filament positive BMSC were found in the brainstemsuggesting that some BMSC differentiated into a neuronalphenotype (Kopen et al., 1999).

Nakano et al. (2001) demonstrated the capacity ofmurine bone marrow cells to differentiate into three dis-tinct glial phenotypes (oligodendrocytes, astrocytes andmicroglia) after direct injection into the corpus striatum ofirradiated mice. Systemic infusion of bone marrow cellsresulted only in the appearance of marrow-derived micro-glia in brain, however, demonstrating the importance ofthe brain microenvironment in providing instructive sig-nals for astroglial and oligodendroglial differentiation. Themarrow cells were tracked by prelabeling with a retroviralvector containing the green fluorescence protein gene.Before labeling, the bone marrow cells were prestimulatedfor 48 hr in fibronectin-coated culture plates and mediacontaining human interleukin-6 (IL-6) and murine stemcell factor. The bone marrow cells were transplanted intoirradiated mice by either systemic infusion or direct injec-tion into the corpus striatum of brain. To identify celltypes, brain sections were stained with specific antibodiesagainst neuronal cell markers: neuron specific enolase(NSE) for neurons, GFAP for astrocytes, carbonic anhy-

drase II (CAII) for oligodendrocytes, and ionized calciumbinding adapter molecule 1 (Iba1) for microglia. Twenty-four weeks after systemic infusion, transplanted cells ex-pressed Iba1 but none of the other brain cell markers.These results are in contrast to those reported by others(Eglitis and Mezey, 1997), who found both microglia andastrocytes in brains of mice when bone marrow cells wereadministered systemically. The population of bone mar-row cells differed in these two studies. Because Nakano etal. (2001) prestimulated the marrow cells with stem cellfactor and IL-6, it is likely the population of cells admin-istered was not the same as that used by Eglitis and Mezey(1997).

DIFFERENTIATION OF BONE MARROWCELLS INTO NEURONS IN VITRO

Studies by Sanchez-Ramos et al. have demonstratedthat a subset of both human and murine bone marrow hasthe capacity to differentiate into neural cells and, in par-ticular, to express markers of early neuron development(Sanchez-Ramos et al., 1998, 2000). BMSC were sepa-rated from whole bone marrow by adherence to polyeth-ylene culture flasks. Proliferation of cells was maintainedby use of epidermal growth factor (EGF). Before induc-tion of differentiation, cultures were enriched in fibronec-tin immunoreactive (ir) cells and depleted of mouse he-matopoietic stem cell (Sca1�) or human hematopoieticstem cells (CD34�). Treatment of the cultures with reti-noic acid (RA) and brain derived neurotrophic factor(BDNF) resulted in decreased number of fibronectin-ircells. This was due to gradual loss of the large, flatfibronectin-ir cells and the appearance of smaller oval cellswith short processes that did not stain with fibronectin(Fig.1, Frame 1). Analysis of BMSC lysates prepared fromcultures treated with either proliferation medium or dif-ferentiation medium demonstrated the presence of nestin,neuron-specific nuclear protein (NeuN) and GFAP.Treatment with RA or RA � BDNF decreased theexpression of nestin protein. Microscopic examination ofthe cultures after immunocytochemical processing re-vealed a small proportion of NeuN-ir and GFAP-ir cells(0.5 and 1%, respectively, of the BMSC cells).

To assess the influence of factors released by devel-oping neural tissues and cell–cell interactions, BMSC ob-tained from transgenic lac-Z mice, which express E. coli�-gal, were co-cultured with fetal mouse mesencepahliccells or neonatal rat forebrain glia cell cultures. Co-culturing of BMSC with fetal mouse midbrain increasedsignificantly the percentage of BMSC that expressedNeuN and GFAP (markers of neurons and astroglia, re-spectively). These results were confirmed in a second set ofexperiments, which utilized BMSC labeled with a fluo-rescent vital stain (Fig.1, Frame 9). The co-culture exper-iments support the hypothesis that cell–cell contact, inaddition to signaling with trophic factors and cytokines,plays an important role in differentiation of these BMSC.The neural cells produced from BMSC in the co-culturesdid not exhibit the morphology of mature neurons or glia,nor did they express microtubule-associated protein

884 Sanchez-Ramos

(MAP)-2, a marker of mature neurons. This may due tothe short duration of incubation (maximum of two weeks)and a slower maturation rate for human-derived cells. Theexpression of NeuN, but not MAP-2, in BMSC-derivedcells is consistent with current knowledge regarding thetimepoints of expression of neuronal proteins: MAP-2 isexpressed at a later developmental stage than NeuN. Im-munohistochemically detectable NeuN protein has beenreported to first appear at developmental timepoints whichcorrespond with the withdrawal of the neuron from thecell cycle or with the initiation of terminal differentiationof the neuron (Mullen et al., 1992). In contrast MAP-1, -2and -3 undergo a number of significant changes duringdevelopment, with the expression of MAP-2 consideredto occur “late,” particularly between 10–20 days in thepostnatal rat pup (Riederer and Matus, 1985).

Whereas Sanchez-Ramos et al. (2000) used RA incombination with growth factors to induce differentiationof BMSC to neural phenotypes (neuron-like cells andglia), others found that �-mercaptoethanol (BME) treat-ment could rapidly induce differentiation into neuron-likecells, but not glial cells (Woodbury et al., 2000; Black andWoodbury, 2001). BMSC (from either rat or human)were propagated in vitro for over 20 passages, attesting to

their proliferative capacity. To induce differentiation toneuron-like cells, the BMSC were transferred to serum-free medium containing 1–10 mM BME. Within 1 hr ofexposure to BME, changes in morphology of some of thecells were apparent. Responsive cells assumed neuronalmorphological traits progressively over the first 3 hr. Cellsexhibited increased expression of the neuronal cytoplasmicmarker NSE within 30 min of treatment. The authorsdocumented a remarkable metamorphosis in the cells overthe next several hours: the cell bodies became increasinglyspherical and refractile and exhibited a typical neuronalperikaryal appearance. BMSC-derived neurons displayeddistinct neuronal morphologies ranging from simple bipo-lar to large, extensive branched multipolar cells (Fig. 1,Frames 10,11). Rare neurons exhibited pyramidal cellmorphologies; neurons elaborating long processes withevident varicosities were more common. To address theissue of long term differentiation, expression of nestin wasmonitored immunocytochemically and by Western blotanalysis of the cultures at 5 hr, 1 day, and 6 days post-differentiation. High levels of nestin protein was seen in asubset of BMSC-derived neural cells at 5 hr, and theproportion of nestin-positive cells decreased with time. By

Š

Fig. 1. Frame 1:Human BMSC grown in presence of RA � BDNF for1 week. Fibronectin� fibroblastic cells are stained red. The unstainedsmall cells (arrows) are neural cell progenitors (with permission fromExp. Neurol., Sanchez-Ramos et al., 2000). Frame 2: Human BMSCtreated with IBMX and db-cAMP for six days differentiate intoneuron-like cells. Phase contrast microscopy (with permission fromBiochem. Biophys. Res. Commun., Deng et al., 2001). Frame 3:Neuronal induction of murine BMSC with noggin showing neuronalphenotype under phase contrast microscopy (with permission fromDifferentiation, Kohyama et al., 2001). Frame 4: Human umbilicalcord blood stromal cells treated with RA � NGF for four days revealMusashi1 immunoreactivity (with permission from Exper. Neurol.,Sanchez-Ramos et al., 2001). Frames 5–8: Neurogenic differentiationof murine BMSC with 5-azacytidine treatment depicts cells expressingTuj-1, Hu, NeuN and GFAP (with permission from Differentiation,Kohyama et al., 2001). Frame 9: Human BMSC treated with RA �BDNF plated on bed of rat fetal midbrain cells express NeuN (greennucleus) and red fluorescent marker (PKH-26) used to permeableBMSC cells (with permission from Exper. Neurol., Sanchez-Ramos etal., 2001). Frames 10,11: Acute differentiation of rat BMSC exposedto �-mercaptoethanol resulting in expression of neuron-specific eno-lase (NSE) within 4 hr. Frame 10 shows heavily stained bipolar neuronsand Frame 11 shows multipolar branching neurons (with permissionfrom J. Neurosci. Res., Woodbury et al., 2000). Frames 12–15:Expression of Neurofilament (Nf-M), tau and NeuN in rat BMSCtreated combination of dimethyl sulfoxide and butylated hydroxyanis-ole. Frame 13 shows effects of preabosrption of the NF-M antibodywith purified NF-M protein to indicate specificity of the Nf-M staining(with permission from J. Neurosci. Res., Woodbury et al., 2000).Frames 16,17: Murine BMSC clone treated with noggin protocol.Frame 16 shows the neuronal morphology in phase contrast and Frame17 documents the influx of calcium ion (red) into neurons in responseto potassium (with permission from Differentiation, Kohyama et al.,2001). Frame 18: Differentiation of rat BMSC treated with5-azacytydine into oligodendrocytes expressing Gal-C (with permissionfrom Differentiation, Kohyama et al., 2001).

Neural Cells Derived From Bone Marrow 885

Day 6 post-induction, there was no detectable nestinexpression in the cells, a finding consistent with ongoingmaturation of neurons. Concomitant with these changes,TrkA, the high affinity nerve growth factor receptor, wasdetectable at 5 hr, and persisted through the 6 days.

Other agents, in addition to BME, were found to beeffective in changing BMSC into a neural phenotype.Dimethyl sulfoxide (DMSO), butylated hydroxyanisole(BHA) and butylated hydroxytoluene (BHT) alone or incombination were found to be effective inducing agents.The most effective treatment was found to be 2% DMSOand 200 �M BHA, which resulted in as high as 78% of thecells expressing NSE. Further analysis also detected otherneuronal markers, including NeuN, NF-m (an interme-diate neurofilament) and tau protein (Fig. 1, Frames 12–15). Interestingly, none of these protocols resulted in thedifferentiation into glial cells, because no GFAP immuno-reactive cells were found.

Kohyama et al. (2001) described two additionalmethods for inducing BMSC to differentiate into neuronsin vitro. One protocol utilized 5-azacytidine (5-Aza-C), ademethylating agent capable of altering gene expression(Holiday, 1996), in a medium containing a combination ofgrowth factors. The induction medium contained5-Aza-C (10 �M) supplemented with the growth factorsNGF, BDNF and NT3. After 96 hr of induction, themedium was replaced with B27-supplemented Dulbecco’sModified Eagle Medium (DMEM)/F12 containing thesame three growth factors. An alternative protocol utilizedthe neural inducer noggin, a diffusible factor that mediatesneural induction during early embryogenesis as well asduring adult neurogenesis by antagonizing bone morpho-genetic protein (BMP) signaling (Smith and Harland,1992). Noggin also converts embryonic stem cells intoprimitive neural stem cells by inhibition of BMP-relatedsignaling (Tropepe et al., 1999). The neurons generatedfrom marrow stroma using either of these two protocolsformed neurites, expressed neuron-specific markers andgenes, and started to respond to depolarizing stimuli asfunctional mature neurons. Among stromal cells, the re-searcher found that isolated mature osteoblasts with strongin vivo osteogenic activity could be converted efficientlyinto functional neurons

Deng et al. (2001) reported that agents that increaseintracellular cAMP levels, such as isobutylmethylxanthine(IBMX) and dibutyryl cAMP (db-cAMP), augmented theproportion of human BMSC that differentiate into a neu-ral cell morphology (Fig. 1, Frame 2). They noted how-ever, that even undifferentiated human BMSC expresssome markers characteristic of neural cells, such asMAP1B, neuron specific tubulin (TuJ-1), NSE and vi-mentin. The treatment with the enhancers of cAMP forsix days increased the expression of NSE and vimentinmRNA without increasing transcription of either MAP1Bor TuJ-1 . There was no detectable expression of NF-m,MAP2, tau, GFAP and myelin basic protein before or afterdifferentiation. These results are somewhat different fromthose obtained in two earlier reports using different culture

conditions (Sanchez-Ramos et al., 1998; Woodbury et al.,2000). The change to a neuron-like morphology wassimilar in all three studies, but the number of neural cellsvaried widely. The results of Deng et al.(2001) weresimilar to those of Woodbury et al. (2000), in that noGFAP was detected, whereas Sanchez-Ramos et al. (1998)observed GFAP expression both before and after differen-tiation.

FUNCTIONAL ACTIVITY OF BONEMARROW-DERIVED NEURONS

Although BMSC have been shown to express pro-teins found typically at various stages of neuronal devel-opment, it was not clear that these neuron-like cells pos-sessed the functional activities of true neurons. So far, onlyone laboratory has published strong evidence for func-tional activity in vitro. Bone marrow-derived neuronalcells were analyzed by whole-cell patch clamp recording(Kohyama et al., 2001). Treatment of BMSC with5-Aza-C led to a decrease in resting membrane potentialto �20 mV on Day 14 and �50 mV on Day 28. A similarresting membrane potential was also observed in neuronsthat served as a positive control. Ionic currents were alsomeasured by the patch clamp method under the voltageclamp condition. With the increase in voltage, rectifyingcurrent was clearly detected, indicating the presence ofvoltage-dependent K� current. The K� channels startedto be expressed concomitantly with the morphologicalchange and increased expression of neuron-specific mark-ers. Moreover, the ability of the bone marrow-derivedneurons to respond to depolarizing stimuli was docu-mented (Kohyama et al., 2001). These cells showed a rapidand reversible calcium increase in response to acetylcho-line, a response characteristic of neurons (Fig. 1, Frames16,17).

NEUROGENIC CELLS MAY BE ISOLATEDFROM UMBLICAL CORD STROMA

Human umbilical cord blood may also harbor cellscapable of differentiation into neural lineages (Sanchez-Ramos et al., 2001). Human umbilical cord blood cellswere plated in culture dishes and incubated withDMEM � 10% fetal calf serum (FCS) for 2 days thenmedia was changed to either RA � NGF or maintained infresh DMEM � FCS for 4–7 days. Microscopic exami-nation of immunostained cultures treated with RA �NGF revealed a heterogeneous mixture of cell types,ranging from large flat epithelioid cells to small spindle-shaped cells with fine branching neuritic processes. Treat-ment of the cultures with RA � NGF resulted in adecrease in total number of cells visualized under phasecontrast microscopy compared to controls treated withDMEM. This treatment also increased the proportion ofthe Musashi-1 immunoreactive cells to 6.2% of the totalcells (Fig.1, Frame 4). RA � NGF treatment also in-creased the proportion of cells (�18.7%) that exhibited�-tubulin III immunoreactivity. Approximately 8% of thecells incubated with DMEM showed �-tubulin III immu-noreactivity. Thirty-four percent of DMEM-treated cul-

886 Sanchez-Ramos

tures were immunoreactive for GFAP, a marker of astro-cytes, but treatment with RA � NGF increased theproportion of GFAP-ir cells to 66.2%. Sixty percent of thecells were immunoreactive for BrdU, indicating that thecells were continuing to proliferate.

Human umbilical cord blood cells prepared as abovewere also transplanted into the developing rat brain, whichis known to provide a conducive environment for devel-opment of neural phenotypes (Zigova et al., 2002). Neo-natal pups (1-day-old) received unilateral injection of a cellsuspension from cord blood cell cultures containing eitherDMEM or RA � NGF into the anterior part of thesubventricular zone (SVZ). One month after transplanta-tion, animals were perfused, brains cryosectioned and im-munocytochemistry was carried out for identification ofneural phenotypes. Approximately 20% of transplantedcells, regardless of their pretreatment, survived within theneonatal brain. The majority of the grafted cells werefound in the SVZ with some dispersion into adjacentcortex and corpus callosum. Cells grafted from theDMEM � FBS cultures were considerably more dispersedthan those from the RA � NGF cultures. Double-labelingwith cell type-specific markers revealed that approxi-mately 2% of the umbilical cord blood-derived cells (fromboth RA � NGF and DMEM-treated cultures) expressedGFAP, and rare cord blood-derived cells (�0.2%) ex-pressed the neuronal marker �-tubulin III. There waslimited migration away from the SVZ but there was nomigration along the rostral migratory pathway to the ol-factory bulb, the usual destination of neural progenitors inthe SVZ. The majority of these cells, regardless of pre-treatment, appeared not to behave like typical neural pro-genitors. This may be a result of the injection of a heter-ogenous cell population already committed to a moredifferentiated state before grafting and a poor survival rateof the human cord blood cells grafted into non-immunosuppressed rat brain. Future work with this modelshould utilize neural progenitors isolated and enrichedfrom umbilical cord and transplanted into an immunosup-pressed animal.

In a study of gene expression using DNA microarraytechnology, umbilical cord blood cells were incubatedunder the same conditions (DMEM � FCS vs. RA �NGF) as described previously (Sanchez-Ramos et al.,2001). Cord blood cells cultured in the presence of RA �NGF exhibited significant changes (�2-fold) in the ex-pression of 322 genes of a total of 12,600 human genesrepresented on the Affymetrix DNA Chip�. The majorityof these genes were not related directly to the process ofneurogenesis; however, at least 20 of these genes could belinked by literature searches to products found in neurons,glia or developing neural cells. For example, the greatestdegree of upregulation (44-fold increase) was seen in themRNA for neurite outgrowth extension protein orpleiotrophin. Several other transcripts associated with earlyneuronal development, such as glypican-4, neuronal pen-traxin II, neuronal PAS1, and neuronal growth associatedprotein 43 (GAP43) all increased significantly. Musashi-1,

the earliest marker of neural precursors, was upregulated1.5-fold. Concomitant with the increased expression ofmarkers indicative of neural development, there was adecrease in expression of many genes associated with de-velopment of blood cell lines. The greatest changes oc-curred in the expression of HLA Class I locus C heavy chain,macrophage receptor MARCO, secreted T-cell activationprotein Attractin (attractin), leucocyte immunoglobulin-likereceptor-8 (LIR-8), thymocyte antigen CD1c, erythropoie-tin receptor and erythropoietin.

These findings suggest that human umbilical cordblood contains cells that can be induced to neural anti-genic and morphological phenotype. Musashi-1 is anRNA-binding protein that has been found in the devel-oping or adult CNS tissues of frogs, birds, rodents, andhumans (Kaneko et al., 2000). The anti-Musashi-1 mono-clonal antibody used in this study was provided by H.Okano and has been shown to react with undifferentiated,proliferative cells of the SVZ in the CNS of all vertebratestested (Kaneko et al., 2000). Both the upregulation and thepost-translational processing of Class-III �-tubulin, one ofthe most specialized tubulins found in neurons (Fanarragaet al., 1999), are believed to be essential throughout neu-ronal differentiation (Laferriere and Brown, 1996; Lafer-riere et al., 1997). The cord blood cultures treated withRA � NGF also increased expression of other genesreported to be specific for neural cells, but we selectedonly a few of them for further study . The increasedexpression of pleiotrophin (neurite outgrowth promotingprotein), an extracellular matrix-associated protein thatenhances axonal growth in perinatal cerebral neurons(Raulo et al., 1992), was independently confirmed byRT-PCR and Western blot analysis Glypican-4 expres-sion was upregulated in the DNA microarray study, butRT-PCR analysis revealed that it was also present in theuntreated cord blood cells. Glypican-4 has been reportedto be expressed in cells immunoreactive for nestin and theD1.1 antigen, markers of neural precursor cells, but hasnot been detected in early postmitotic or fully differenti-ated neurons (Hagihara et al., 2000). Necdin mRNA, amarker specific for neurons (Taniura et al., 1998; Yo-shikawa, 2000), was detected in the RT-PCR analysis intreated and untreated cells, and its protein expression(NeuN) was shown to be increased by 19% in the Westernblot analysis.

GENE EXPRESSION IN NEURALPROGENITOR AND HEMATOPOIETICCELLS DIFFERENTIATION: CLUES TO

META-DIFFERENTIATION?The conversion of cells of mesodermal origin (os-

teoblasts) into cells that normally have ectodermal origin(neural cells) has been termed “meta-differentiation” (Ko-hyama et al., 2001). The mechanism for meta-differentiation may involve at least two basic processes,operating alone or in concert: 1) a reprogramming of thegene expression profile of a differentiated cell into that ofa pluripotent cell, and 2) proliferation and differentiationof a pluripotent progenitor/stem cell already harbored in

Neural Cells Derived From Bone Marrow 887

the BMSC. The reprogramming of a differentiated cellinto a pluripotent one has been termed transdifferentia-tion, not to be confused with dedifferentiation, a processthat also results in reversion of a mature cell into a moreprimitive state as a preliminary to neoplastic transforma-tion. The best example of transdifferentiation is the repro-gramming that occurs in a somatic nucleus transferred intothe environment of an enucleated egg (as in reproductivecloning). The cytoplasm of the egg contains instructivesignals that reset the old program of the epithelial cellnucleus into that of a fresh totipotent cell capable ofgenerating a complete organism. Little is known about thesignaling molecules in egg cytoplasm responsible for thisprocess of reprogramming. More is known about com-pounds used to induce meta-differentiation of bone mar-row cells into neurons, such as the demethylating agent5-Aza-C or RA in conjunction with growth factors(Umezawa et al., 1992; Holiday, 1996). These moleculeshave direct and indirect actions on gene transcription,supporting the concept that reprogramming the genome isthe key to understanding meta-differentiation.

Clues to the molecular mechanism responsible fortransdifferentiation can be found in the systematic study ofgene expression profiles that orchestrate differentiation ofstem cells into specific phenotypes. Gene expression stud-ies in stem cell populations have been facilitated by com-bining a powerful genetic subtraction technique, repre-sentational difference analysis (RDA), with cDNAmicroarray analysis, and validated by in situ hybridizationto see where the gene(s) are expressed (Geschwind et al.,2001). This method overcomes the limitation of workingwith heterogeneous populations containing not only stemcells, but also numerous committed progenitor cells anddifferentiated cells. An RDA subtraction was performed inwhich cDNA from neural stem cell cultures that had beendifferentiated for 24 hr was subtracted from cDNA of sistercultures that were maintained as stem/progenitor cultures(the neurosphere condition). This analysis identifiedknown and novel genes enriched in neural progenitorcultures. Using in situ hybridization technique, manygenes were found to be expressed preferentially in thegerminal regions of brain (the ventricular and subventricu-lar zone). Several genes were enriched in hematopoieticstem cells (HSC) suggesting an overlap of gene expressionin neural and hematopoietic progenitors (Geschwind etal., 2001). In a converse study, a large set of HSC geneswere found to be expressed in mouse neurospheres, apopulation containing neural progenitor cells (Terskikh etal., 2001). Many of these are genes known to be involvedin cell cycling, DNA repair and signaling machinery.Some of these genes are candidate markers for HSC andneural progenitors, useful for future experiments to iden-tify neural progenitors at the earliest stages. Could it bethat expression of some early neural proteins in bonemarrow derived cells is simply an epi-phenomenon, re-flecting shared genetic programs characteristic of all stem/progenitor cells? This would suggest that the bone marrowcells expressing a few neural proteins are really marrow

cells artificially caught in early stages of differentiation, andare not truly neurons. There is compelling evidence forthe conversion of osteoblasts into mature neurons, how-ever, complete with electrophysiologic function typical ofneurons (Kohyama et al., 2001). The gene expressionprofiles of stem cells, regardless of their pedigree, does shedlight on the molecular basis of “stemness.” The genesexpressed in both HSC and neural stem cells very likelyevolved to participate in fundamental stem cell functions,including mechanisms for regulating proliferation, differ-entiation and protection of the genome during lifelong cellrenewal (Terskikh et al., 2001). Although the results fromthese gene expression studies provide some clues to themolecular basis for transdifferentiation, the mechanismremains poorly understood. With the advances in cDNAmicroarray technology and functional genomics, the toolsare now available to address the problem systematically.

MIGRATION AND IN SITUDIFFERENTIATION OF BONE MARROWCELLS INTO A NEURONAL PHENOTYPE

AFTER TRANSPLANTATIONAn interesting property of BMSC is their proclivity

to migrate. Two elegant studies have shown that trans-planted adult bone marrow cells can migrate from systemiccirculation to the brain, where they undergo transdiffer-entiation into neurons as well as microglia and astroglia.Adult bone marrow cells from male mice were adminis-tered systemically to females of a mouse strain (PU.1knockouts) that lacks macrophages, neutrophils, mast cells,osteoclasts and B- and T-cells at birth (Mezey et al., 2000).These animals require a bone marrow transplant within48 hr of birth if they are to survive. Within 24 hr afterbirth, PU.1 homozygous recipients were given intraperi-toneal injections of unprocessed bone marrow cell suspen-sions (containing 107 cells) from wild-type male mice.Between 1–4 months after transplantation, marrow-derived cells were present in the brains of all the trans-planted mice examined. Between 2.3–4.6% of all cells (allidentifiable nuclei) were Y-chromosome positive (derivedfrom donor bone marrow). The Y-chromosome bearingcells were evenly distributed throughout different brainregions. The Y-chromosome was present in 0.3–2.3% ofneurons, marked by immunoreactivity for neuron specificnuclear protein (NeuN). In the brains of transplantedfemale mice, all the Y-chromosome� neuronal nuclei alsoexpressed NSE. These studies demonstrate that bone mar-row cells administered systemically can migrate into thebrain and differentiate into cells that express neuron-specific antigens.

In a similar study, another group found that bonemarrow cells infused into irradiated mice migrated into thebrain and differentiated into cells that expressed neuronalantigens (Brazelton et al., 2000). Adult marrow was har-vested from transgenic mice that ubiquitously express en-hanced green fluorescent protein (GFP). GFP-expressing(GFPpos) bone marrow was administered by tail vein(6 million cells/recipient) into lethally irradiated, isogenicmice. Brains harvested several months after the transplant

888 Sanchez-Ramos

and examined by light microscopy revealed the presenceof GFPpos cells throughout the brain, including the olfac-tory bulb, hippocampus, cortical areas and cerebellum.Examination of dissociated brain and bone marrow cellsfrom the recipients revealed that essentially all of theGFPpos cells that engrafted in the host bone marrow alsoexpressed CD45 (surface marker of all nucleated matureblood lineages). A significant subset (up to 20%) of theGFPpos cells that engrafted in the brain, however, lackedboth CD45 and CD11b (surface markers expressed by allmyelomonocytic cells). These findings suggested that ex-posure to a brain microenvironment led a subpopulationof bone marrow-derived cells to acquire novel pheno-types. Using confocal microscopy, the researchers deter-mined that individual cells co-expressed GFP and neuron-specific antigens. The olfactory bulb was selected forindepth quantification and revealed that 0.2–0.3% of thetotal number of neurons were derived from bone marrowby 8–12 weeks after transplantation. A substantial propor-tion of marrow-derived cells co-expressed multipleneuron-specific gene products including two neuronalproteins, 200-kD neurofilament (Nf-H) and Class III�-tubulin, but did not express the glial cell marker GFAP.

SPONTANEOUS CELL FUSION: APOTENTIAL EXPLANATION FOR THE

CHANGE IN PHENOTYPE AFTERTRANSPLANTATION?

Two recent independent studies suggest that trans-differentiation, or conversion to a neural phenotype, aftertransplantation of Y-chromosome or GFP-labeled BMSCmay be due to a cell fusion phenomenon. These reportshave raised some doubt concerning the plasticity of adulttissue-derived stem cells. One of the studies demonstratedthat adult murine bone marrow cells can fuse spontane-ously with embryonic stem cells when co-cultured inmedia containing interleukin 3 (Terada et al., 2002). Theresulting hybrid cells exhibited multipotentiality, an ob-servation that mimics the phenotypic conversion ofBMSC to unexpected cell lineages. The other studyshowed mouse neural progenitor cells, when co-culturedwith pluripotent embryonic stem cells, gave rise to mul-tipotent cells that expressed genetic markers of the neuralcell. Ostensibly it would appear that the neural cells de-differentiated into a multipotent cell line or transdifferen-tiated into unexpected phenotypes, but the investigatorswere able to demonstrate these cells were tetraploid hy-brids with full pluripotent character (Ying et al., 2002).The authors of these two studies suggest that cell fusionmight be responsible for the observations attributed to anintrinsic plasticity of adult tissue stem cells. This is certainlya possibility in studies where adult cells are co-incubatedwith multipotent stem cells in vitro. It might also occurwhen adult BMSC are grafted into adult brain and there isfusion of the donor marrow cells with neural progenitorslocated in neurogenic zones (SVZ). The results of five invitro studies cannot be explained by cell fusion, however,because adult tissue-derived BMSC were not co-culturedwith any cells (Table I). Only one in vitro study reviewed

here utilized co-cultures. In the very first demonstration ofneural cells generated from BMSC in vitro, the low fre-quency of neuron-like cells generated from BMSC inmonoculture was increased by co-culturing the BMSCwith fetal brain tissue(Sanchez-Ramos et al., 2000), Al-though cell fusion may be the mechanism for enhancedconversion of phenotype, unknown humoral factors elab-orated by the fetal cells and cell–cell contact may also haveplayed a role in the transdifferentiation process. In anycase, the spontaneous cell fusion phenomenon serves tocaution researchers who study plasticity of adult stem cells.Future work involving co-culture or transplant studies willneed to test for the possibility of cell fusion and generationof cell hybrids.

ISOLATION AND CHARACTERIZATION OFTHE SUBPOPULATIONS OF BONE

MARROW CELLS CAPABLE OFDIFFERENTIATION INTO

A NEURAL LINEAGEThe cell, or subpopulation of cells, that is the source

of neurons and glia or that actually enters the brain anddifferentiates in situ into neurons remains unclear, becausemany studies used heterogeneous or poorly characterizedpopulations of bone marrow cells. Some investigatorsmade an effort to eliminate hematopoietic precursors fromthe marrow cells before inducing differentiation in vitro orbefore transplantation. Hematopoeitic stem cells (CD34�

cells from human bone marrow or Sca1� cells from mousebone marrow) were separated by immunomagnetic beadsorting before preparing stromal cell cultures (Sanchez-Ramos et al., 2000). Other researchers utilized fluorescentcell sorting with flow cytometry of bone marrow stromalcells after the first passage to demonstrate that the BMSCwere negative for CD11b and CD45, surface markersassociated with lympohematopoietic cells (Black andWoodbury, 2001). Although neural cells were differenti-ated from cultures depleted of hematopoietic progenitorcells in these two studies, the BMSC cultures consisted ofheterogenous cell types. From these studies it was notpossible to determine whether a primitive multipotentstem cell resides in adult bone marrow or whether atransdifferentiation process occurred. Subcloning by lim-iting dilution provides a better approach to this problembecause it is assumed that all the cells are derived from asingle multipotent stem cell that resides in adult bonemarrow. Several clones of BMSC were generated fromfemale mice by limiting dilution (Kohyama et al., 2001).The clones could differentiate into osteogenic, adipogenicand myogenic lineages under specific culture conditionsreported earlier by other investigators (Pittenger et al.,1999). Without any treatment, the BMSC clones exhib-ited a fibroblastic appearance. All three clones exhibitedhigh alkaline phosphatase activity, indicating they had anosteogenic potential. All three of the clones also expressedthe following surface markers: Sca1, (but not c-kit), CD29(Integrin1), CD140 (platelet-derived growth factor recep-tor �), CD44 (Pgp-1/Ly-24), and Ly6c (marker of osteo-blasts). Neural differentiation was induced by incubation

Neural Cells Derived From Bone Marrow 889

with 5-Aza-C (10 �M) for 96 hr and continued incuba-tion for NGF/NT3/BDNF, as described earlier. TheBMSC changed from a fibroblastic appearance to neuron-like cells. In two of the clones, 20% of the cells formedneurite-like processes and exhibited a neuron-like mor-phology by Day 9 after treatment. Immunocytochemistryrevealed the cells were positive for neuronal markers in-cluding NeuN, Tuj1, and Hu. These cultures also werepositive for markers of astroglia (GFAP) and oligodendro-cytes (Gal-C). The number of positive cells differedamong the three cell lines, suggesting that each cell linecontained several committed progenitors that indepen-dently give rise to neurons, astrocytes and oligodendro-cytes. The fact that all three clones expressed alkalinephosphatase (a marker of osteogenic potential) and yetcould be induced to differentiate into neural cells aftertreatment with 5-Aza-C, a methylation modifier, lendssupport to the transdifferentiation process. This hypothesissuggests that reprogramming of the nucleus of osteoblasticprogenitors is responsible for the activation of neuroecto-dermal genetic programs. Lineage interconversion be-tween distinct adult committed progenitors is unexpectedbut not impossible. Expression of neurogenic phenotypeshave been reported in bone-originated sarcomas (Ewingsarcoma), an uncommon neoplasm of bone and extraosse-ous tissue (Sugimoto et al., 1997; Gardner et al., 1998).These neoplastic sarcoma cells occasionally express neuro-ectodermal markers, such as NSE, neurofilament andnerve growth factor receptor, suggesting a relationship ofEwing sarcoma to neuroectodermal tumors (Sugimoto etal., 1997; Chung et al., 1998; Gardner et al., 1998).

Another approach for producing a homogeneouspopulation of bone marrow-derived cells capable of gen-erating neural lineages was described by the laboratory ofVerfaillie (Reyes et al., 2002). Human bone marrowmononuclear cells obtained by Ficoll-Pague density gra-dient centrifugation were depleted of CD45� andglycophorin-A� cells, (cells of hematopoeitic lineage) byimmunomagnetic sorting. After two depletion steps , themononuclear cells were greater than 99.5% free ofCD45�, GlyA� cells. The remaining cells (0.5% of themononuclear fraction), were propagated for at least 20doublings, resulting in a homogeneous population of cellstermed multipotent adult progenitor cells (MAPC). By10–15 cell doublings, MAPC did not express CD10,CD31, CD34, CD36, CD38, CD50, CD62E, CD106,CD117, HIP12, fibroblast antigen, HLA-DR, class IHLA, CD45, Tie, or Tek. These cells did not changemorphology and phenotype for up to 70 doublings. TheMAPCs were shown to differentiate under specific cultureconditions to mesenchymal cell types (cartilage, bone,fibroblasts, and adipocytes), and to cells of almost all othermesodermal lineages (skeletal, smooth and cardiac myo-blasts, and endothelial cells). Most interesting, the MAPCwere induced to differentiate to cells of neuroectodermallineage, including �-tubulin-III, neurofilament, NSE andglutamate-positive neurons, GFAP� astrocytes and galac-tocerebroside� oligodendrocytes (Reyes and Verfaillie,

2001). Based on the number of steps and passages used inthe enrichment procedure, the frequency of the MAPCwas calculated to be extremely low (1 in 107 or 108). Theirorigin remains speculative, but it has been suggested thatthey are descendants from primordial germ cells, whosefunction remains unknown (Reyes and Verfaillie, 2001). Itis also possible, however, that the frequent passaging re-sulted in the creation of a new cell line, through a poorlyunderstood process of dedifferentiation. Verfaillie notesthat the MAPC cultures stop growing, however, whenthey become confluent and hence do not behave like aneoplastic cell line (Reyes and Verfaillie, 2001).

BONE MARROW STROMAL CELLS FORTHERAPY OF STROKE, TRAUMA,

AND PARKINSON DISEASEAnticipating the therapeutic potential of these fun-

damental observations, bone marrow cells have beenshown to hasten recovery of neurologic deficits in rodentmodels of stroke, brain and spinal cord trauma and Par-kinson Disease. Researchers have demonstrated that directintracerebral grafting and intravenous infusion of BMSCare effective in hastening recovery from the neurologicaldeficit induced by middle cerebral artery occlusion (Li etal., 2000; Chen et al., 2001a). One of the first reportsdescribed direct transplantation of adult BMSC, prelabeledwith BrdU, into the striatum after embolic middle cerebralartery occlusion (Li et al., 2000). The mice were killed28 days after stroke. BrdU-reactive cells survived andmigrated a distance of approximately 2.2 mm from thegrafting areas toward the ischemic areas. NeuN was co-expressed in 1% of BrdU stained cells and the astrocyticspecific protein GFAP in 8% of the BrdU stained cells.Functional recovery from a rotarod test and modifiedneurologic severity score tests (including motor, sensory,and reflex) were improved significantly in the mice re-ceiving the BMSC graft. Similar functional recovery wasachieved after intravenous infusion of BMSC into rats aftera middle cerebral artery embolic stroke (Chen et al.,2001a). In both of these studies there were few marrow-derived cells that expressed neuron-specific markers, andthe size of the infarct was not altered in animals thatreceived the BMSC compared to stroked animals withoutthe BMSC. It is difficult to explain the mechanism ofneurologic recovery other than to suggest that these cellselaborated a number of trophic factors and cytokines thatpromote recovery in the host. In support of this hypoth-esis, Chen et al. (2000) found that a composite graft offresh BMSC along with brain-derived neurotrophic factor(BDNF), transplanted into the ischemic boundary zone ofrat brain, improved survival and differentiation of theBMSC. This combined treatment also hastened functionalrecovery after middle cerebral artery occlusion. Thesestudies show clearly a therapeutic benefit in a strokemodel, but the correlation between the differentiation ofneural cells and recovery is poor. Other characteristics ofthese cells that may play a role in functional recovery fromstroke remain to be explored.

890 Sanchez-Ramos

Transplantation of BMSC into the spinal cord after acontusion injury was also reported to enhance recoverybased on standardized assessments (Chopp et al., 2000).Rats were subjected to a weight-driven implant injury.BMSC or PBS was injected into the spinal cord one weekafter injury. Sections of tissue were analyzed by double-labeled immunohistochemistry for BMSC identification.Functional outcome measurements were carried outweekly, up to 5 weeks post-injury. The data showed asignificant improvement in functional outcome in animalstreated with BMSC transplantation compared to controlanimals. Scattered cells derived from BMSC expressedneural protein markers, but like the stroke studies, it isunlikely that reconstruction of injured spinal cord bydifferentiating marrow cells is responsible for the improve-ment. The enhanced recovery, according to speculationby the authors, is most likely mediated by humoral factorsreleased by marrow cells.

Treatment of traumatic brain injury by intravenousinfusion or direct intracerebral grafting of BMSC alsoenhanced recovery from the neurologic deficit (Mah-mood, 2001; Mahmood et al., 2001). More specifically,there was a significant improvement in motor function, 14and 28 days after transplantation, in the transplanted ratscompared to control rats. Histological examination of therat brains revealed that marrow-derived cells survived,proliferated, and migrated toward the injury site. A smallproportion of the BrdU-labeled BMSC cells expressedneuronal (NeuN) and glial (GFAP) markers. Like theother studies in this series, it is unlikely that the differen-tiation of BMSC into neuron-like cells and glia explainsthe recovery.

The therapeutic potential of BMSC for the treat-ment of Parkinson disease was given an impetus by arecent publication from the same group of researchers (Liet al., 2001). BMSC prelabeled with BrdU were graftedinto the striatum of MPTP-treated mice. The graftedMPTP-treated mice exhibited a significant improvementon the rotarod test at 35 days after transplant, compared tonongrafted controls. Immunohistochemistry revealedBrdU reactive cells in the striatum of the grafted MPTP-treated mice at least four weeks after transplantation. Scat-tered BrdU-reactive cells expressed tyrosine hydroxylase(TH) immunoreactivity. Although the BMSC injectedintrastriatally survive, express TH immunoreactivity, andpromote some functional recovery, much more work isrequired to understand the mechanism for this recovery. Itis not known whether the grafted cells increase productionof dopamine or whether other processes, such as thesecretion of neurotrophic factors by the marrow-derivedcells, mediate the improvement in motor function.

CONCLUSIONThe conversion of bone marrow or umbilical cord

blood cells into neurons and glia may be explained byseveral mechanisms that are not mutually exclusive andmay act in concert: 1) The stem cells found in adult tissuesare true multipotent stem cells that arrive in the adult tissueearly in development (or perhaps migrate to the organ later

in development), but retain “stemness” (self-renewal,multipotency) in the adult tissue throughout life; and 2)tissue-specific progenitor cells, with limited differentiationpotential, normally designed to replace cells specific to theorgan of residence, have the capacity under experimentalconditions to transdifferentiate into cells characteristic ofother tissues and organs. This latter explanation implies agenetic reprogramming of the nucleus of the progenitorby instructive molecular signals, similar to what occurswhen a mature somatic cell nucleus is inserted into an eggto generate a totipotent cell capable of producing all cellsthat make up a complete organism (as in reproductivecloning). Recent in vitro observations point out a con-founding phenomenon of fusion of adult tissue cells withembryonic stem cells that should raise a flag of cautionwhen interpreting transdifferentiation experiments. With-out understanding completely the molecular mechanismsresponsible for adult tissue stem cell plasticity, it is stillpossible to move forward in the translation of these find-ings to clinical applications. The precedent for using bonemarrow and umbilical cord blood to replace bone marrowof patients with leukemia is well established, providingadditional impetus for exploring bone marrow transplan-tation in the treatment of neurodegenerative diseases,trauma and stroke.

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