Banfi 2000 Experimental-Hematology

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Experimental Hematology 28 (2000) 707715

0301-472X/00 $see front matter. Copyright 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc.

PII S0301-472X(00)00160-0

Proliferation kinetics and differentiationpotential of ex vivo expanded human bone marrow

stromal cells: Implications for their use in cell therapy

Andrea Banfia,b,c, Anita Muragliaa, Beatrice Dozina,b,Maddalena Mastrogiacomoa,c, Ranieri Canceddaa,b,c, and Rodolfo Quartoa,b

aCentro di Biotecnologie Avanzate, Genova, Italy;bIstituto Nazionale per la Ricerca sul Cancro, Genova, Italy; cDipartimento di Oncologia, Biologiae Genetica, Universita di Genova, Italy

(Received 4 January 2000; revised 12 February 2000; accepted 22 February 2000)

Objective. Bone marrow stromal cells (BMSC) are an attractive target for novel strategies in

the gene/cell therapy of hematologic and skeletal pathologies, involving BMSC in vitro expan-

sion/transfection and reinfusion. We investigated the effects of in vitro expansion on BMSCpluripotentiality, proliferative ability, and bone-forming efficiency in vivo.

Materials and Methods. BMSC from three marrow donors were cultured to determine their

growth kinetics. At each passage, their differentiation potential was verified by culture in in-

ductive media and staining with alizarin red, alcian blue, or Sudan black, and by immu-

nostaining for osteocalcin or collagen II. First passage cells were compared to fresh marrow

for their bone-forming efficiency in vivo. Stromal cell clones were isolated from five donors

and characterized for their multidifferentiation ability. The lifespan and differentiation kinet-

ics of five of these clones were determined.

Results. After the first passage, BMSC had a markedly diminish proliferation rate and gradu-

ally lost their multiple differentiation potential. Their bone-forming efficiency in vivo was re-

duced by about 36 times at first confluence as compared to fresh bone marrow. Experiments

on the clones yielded comparable results.

Conclusions. Culture expansion causes BMSC to gradually lose their early progenitor proper-ties. Both the duration and the conditions of culture could be crucial to successful clinical use

of these cells and must be considered when designing novel therapeutic strategies involving

stromal mesenchymal progenitor manipulation and reinfusion. 2000 International Society

for Experimental Hematology. Published by Elsevier Science Inc.

Keywords: ChondrogenesisOsteogenesisAdipogenesisBone marrow stromal cellsCell

therapy

Introduction

The bone marrow stromal system is defined as the connec-

tive tissue elements providing structural and functional sup-

port for hemopoiesis [1,2] and includes various cell types:bone marrow fibroblastsreticular cells, adipocytes, osteo-

blasts, macrophages, and endothelial cells. It has long been

recognized that the fibroblastoid component of the stromal

system contains a rich reserve of osteogenic progenitors that

also are capable of transferring the hemopoietic microenvi-

ronment [35] both in vitro [6] and in vivo [7,8].

A mesenchymal stem cell (MSC) is defined as a pluripo-

tent cell capable of replicating extensively and self-main-taining and whose progeny eventually gives rise to skeletal

tissues: cartilage, bone, tendon, ligament, and marrow

stroma [5]. By definition, such cells exist during embryo-

genesis, and it is the object of current research to determine

whether these cells persist during adult life.

Bone marrow stromal cells (BMSC) can be cultivated in

vitro and contain progenitors capable of pluridifferentiating

into the mesenchymal lineages of bone, cartilage, fat, mus-

cle, and other connective tissues [3,5,911]; therefore, they

are an interesting target for use in cell and gene therapy.

Offprint requests to: Rodolfo Quarto, M.D., Laboratorio di Differenzia-

mento Cellulare, Centro di Biotecnologie Avanzate/Istituto Nazionale per

la Ricerca sul Cancro, L.go R. Benzi n. 10, 16132 Genova, Italy; E-mail:

[email protected]


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708 A. Banfi et al. /Experimental Hematology 28 (2000) 707715

BMSC attractiveness is based on the ease with which they

can be isolated from the patients marrow, expanded many-

fold in vitro, manipulated, and reinfused to the same patient.

In this setting, BMSC can have many hematologically rele-

vant applications. They are being investigated as vehicles

for gene therapy of hemophilia B [12]. Several authors have

shown that the bone marrow microenvironment is heavily

and permanently damaged by bone marrow transplantation

(BMT) conditioning regimens [1321]. A preliminary re-

port has claimed that autologous ex vivo expanded BMSC

accelerated hemopoietic recovery after high-dose chemo-

therapy with peripheral blood progenitor cells (PBPC) res-

cue in breast cancer patients [22]. BMSC can be used in

BMT as a therapy for skeletal pathologies [23,24] and has

been applied to cases of osteogenesis imperfecta [25,26].

The use of BMSC in such therapeutic strategies relies on

the ability of BMSC to proliferate readily and give rise to a

differentiated progeny that can substitute for the diseased

counterpart. It is of interest to determine whether, after the

ex vivo expansion necessary before their therapeutic manip-ulation and reinfusion, these cells possess stem features. In

this study, we investigated the stem cell characteristics of ex

vivo expanded human BMSC by asking the following ques-

tions: (1) Do BMSC maintain a constant doubling time dur-

ing their in vitro culture? (2) Do BMSC maintain their own

multilineage differentiation potential after a number of mi-

totic divisions? (3) Do BMSC maintain a constant effi-

ciency in their bone-forming ability in vivo? (4) Do clones

derived from individual colony-forming unit-fibroblasts

(CFU-F) display a multilineage differentiation potential and

do they maintain it throughout their lifespan?

To this purpose, we investigated the in vitro growth ki-netics of BMSC from three different donors in the primary

culture and up to the fourth passage. We analyzed their dif-

ferentiation potential at each passage to monitor the mainte-

nance of pluripotentiality during mitotic expansion. To fur-

ther test this point, we compared the in vivo bone-forming

efficiency of first passage BMSC to that of fresh bone mar-

row. In both experiments we found that, as a population, ex-

panded BMSC progressively lost their differentiation poten-

tial.

The nearest approximation to the putative MSC that can

be studied is the CFU-F. We isolated, expanded, and char-

acterized 80 nonimmortalized clones originating from sin-

gle CFU-F of human bone marrow. Their osteo-chondro-adipogenic potential was assessed by in vitro assays. All

BMSC clones analyzed were able to differentiate in vitro

into the osteogenic lineage and about one-third displayed

osteo-chondro-adipogenic potential. The lifespan was deter-

mined and the differentiation potential was assessed at in-

creasing cell doublings in a number of clones. The results

paralleled those of BMSC cultures.

This study provides evidence that, with extended culture

in the conditions currently in use, human BMSC display a

tendency to lose their multipotentiality, proliferation poten-

tial, and in vivo bone-forming efficiency. The question

whether the graduality of this process could make a limited

expansion compatible with successful clinical use awaits in

vivo studies. Nevertheless, the effect of in vitro expansion

on the stem cell features of BMSC cultures need to be con-

sidered when designing strategies for use of BMSC in gene

and cell therapy.

Materials and methods

Cell culture and BMSC growth kinetics determination

Stromal cells were obtained from iliac crest marrow aspirates from

healthy donors for BMT procedures carried out in S. Martino Hos-

pital, Genova, Italy, and G. Gaslini Pediatric Hospital, Genova, It-

aly. Donor ages ranged between 5 months and 30 years; all donors

were white Caucasians. Informed consent was obtained from all

donors, and all procedures were approved by the institutional ethi-

cal committee. BMSC cultures were performed essentially as pre-

viously described [13], except that they were always carried out

with medium supplemented with 1 ng/ML human recombinant fi-broblast growth factor 2 (FGF-2) (Austral Biologicals, San Ra-

mon, CA). After 2 weeks, CFU-F number was determined as de-

scribed [13]. Confluent cultures were detached with 0.05%

trypsin-0.01% EDTA (Sigma, St. Louis, MO). Cells were replated

for the in vitro differentiation experiments and for determination

of BMSC growth kinetics. At the first passage, BMSC were re-

plated at a concentration of 5 105 cells in one 10-cm Petri dish.

When subconfluent, this plate was successively split 1:2 unit eight

dishes were obtained. At this point, cells again were detached,

pooled, counted, and analyzed for differentiation potential. The

same procedure was repeated for five passages, and the cumulative

cell doublings of the populations were plotted against time in cul-

ture to determine the growth kinetics of BMSC expansion.

Cell cloning

Marrow samples were used to obtain clones by limiting dilution.

Briefly, 2 105 nucleated marrow cells were plated in each of four

to five 96-well plates for each marrow sample. Cultures were per-

formed in standard medium in the presence of FGF-2 and exam-

ined daily for the appearance of stromal colonies. Wells containing

more than one colony were not considered. All clones that reached

confluence after passaging into one well of a 24-well plate were

trypsinized and each of them replated in four wells (replicas). Of

the four replicas of each clone, three were stimulated for the differ-

entiation experiments when cells reached confluence and one was

frozen to be kept as stock.

In vitro differentiation of BMSC cultures and clones

BMSC potential to differentiate into the chondrogenic, osteogenic

and adipogenic lineages was verified at the level of primary cul-

tures and at each passage for four passages. Clones were analyzed

at the stage of replicas and at the following passages during their

lifespan. In all cases, cells were enzymatically detached from cul-

ture dishes on reaching 90% confluence and plated at 5 104

cells/cm2 in 24-well culture plates. Some dishes were not replated

in the wells, but instead were stimulated under the same conditions

and then used for RNA extraction and reverse transcriptase-poly-

merase chain reaction (RT PCR) analysis. Each well or dish was

kept in complete medium until confluence was reached. Cultures


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A. Banfi et al. /Experimental Hematology 28 (2000) 707715 709

were stimulated with the appropriate differentiating medium ac-

cording to the following conditions:

Chondrogenic differentiation: cultures were stimulated for 1

week in standard medium (F-12 with 10% fetal calf serum

[FCS] and without FGF-2) supplemented with 50 g/mL

ascorbic acid and 1 ng/mL human recombinant transforming

growth factor 1 (TGF-1; Austral Biologicals, San Ramon,

CA). Type II collagen expression was revealed by immu-nostaining with the monoclonal antibody CIICI (Develop-

mental Studies Hybridoma Bank) and the deposition of pro-

teoglycans was revealed by alcian blue staining.

Osteogenic differentiation: cultures were stimulated for 2

weeks in standard medium supplemented with 50 g/mL

ascorbic acid, 1.5 mg/mL -glycerophosphate, and 108M

dexamethasone. Osteocalcin expression was revealed by im-

munostaining with an antiserum against bovine osteocalcin

kindly provided by Dr. Simon Robbins, and the presence of

calcium deposits was revealed by von Kossa and alizarin S

staining.

Adipogenic differentiation: cultures were stimulated for 3

weeks in F12 medium supplemented with 1% FCS, 107

Mdexamethasone, and 6 ng/mL insulin. Lipid droplets were re-

vealed by staining with Sudan black IV.

As controls, the following culture conditions were used: (a)

proliferating unstimulated cells expanded with FGF-2 up to con-

fluence (basal phenotype); and (b) confluent cultures kept in stan-

dard medium for the same period of time as the corresponding

differentiation protocols, but without the stimulating factors (sponta-

neous over-time phenotype).

The cultures were scored as positive, borderline, or negative.

For adipogenesis, scores were defined as follows: negative no

more than a single adipocyte in from a total of three random mi-

croscopic fields; borderline one or two adipocytes in each of at

least two of three random microscopic fields; positive at leastthree adipocytes in each of at least two of three random micro-

scopic fields. For osteogenesis and chondrogenesis, we quantified

alizarin S and alcian blue staining, respectively. Images of the

stained wells were acquired with a Cohu black-and-white camera

and analyzed with the NIH-Image 1.60 public domain software.

We scored the average of duplicate wells for each condition. Back-

ground was calculated from empty wells and subtracted from all

experimental readings. Differentiation units were calculated as the

fold increase in staining induced by the differentiation schedule as

compared with the negative control. Cultures were scored as fol-

lows: negative less than 1.2 differentiation units; borderline

between 1.2 and 2 differentiation units; positive more than 2 dif-

ferentiation units.

In vivo bone-forming efficiency

In vivo ectopic bone formation was assayed according to a proto-

col previously optimized in our laboratory [27]. Briefly, on reach-

ing their first confluence, 2.5 105 BMSC were loaded on a

highly porous hydroxyapatite support and implanted subcutane-

ously on the back of nude CD-1 nu/nu mice purchased form

Charles River Italia (Calco, Italy).

When fresh bone marrow was used, nucleated cells were iso-

lated by Ficoll gradient centrifugation and counted, and each cube

was loaded with the appropriate number of cells. Preliminary ex-

periments were carried out to determine the number of fresh mar-

row nucleated cells, of which the majority are hemopoietic progen-

itors, that produce a bone formation equivalent to the standard

2.5 105 expanded BMSC.

The fresh marrow content of CFU-F was determined as de-

scribed earlier. For BMSC, and additional colony-forming effi-

ciency experiment was performed on reaching the first confluence,

in parallel with the in vivo implants, by plating 103 BMSC per 10-

cm Petri dish. After 2 weeks of culture, the plates were fixed andstained, and the colonies were counted as for the fresh marrow.

Animals were sacrificed 8 weeks after implantation, and the grafts

removed and processed for histologic analysis as previously de-

scribed [27].

Clone lifespan

Five clones were selected after characterization at the stage of rep-

licas for determination of their lifespan and maintenance of differ-

entiation ability: three were tripontential (OCA [osteo-chondro-

adipogenic]) and two were bipotential (OC [osteo-chondrogenic])

at this stage. The frozen replica was thawed and plated in a 6-cm

Petri dish. When it reached confluence, cells were trypsinized,

counted for determination of cell doublings, and split into four

equal parts: three were plated each in a well of a 24-well plate andstimulated for differentiation to the three lineages as described ear-

lier, and the fourth was replated in a 6-cm Petri dish to continue

expansion. For these experiments, osteogenesis and chondrogene-

sis were assayed as osteocalcin and collagen II expression as deter-

mined by immunohistochemistry. The results could not be quanti-

fied and were only scored as positive or negative as compared to

the negative control.

Semiquantitative RT-PCR measurements

Total RNA was extracted from BMSC and control tissues (human

articular cartilage, bone, and adipose tissue) with the guanidinium

isothiocyanate procedure by Chomczynski and Sacchi [28]. Semi-

quantitative RT-PCR was performed using the GeneAmp RNAPCR kit from Perkin Elmer. For each RNA sample, a master RT

reaction was performed with 2 g total RNA in a 40 L mixture

containing 1 PCR buffer from the provider, 5 mM MgCl2, 1 mM

each of dCTP, dATP, dGTP, and dTTP, 1 U/

L of RNAse inhibi-

tor, 250 pmol of random examer primers, and 2.5 U/L of MuLV

RT. Reaction time was 1 hour at 42C. Each master cDNA product

was divided into two equal parts that were used for PCR amplifica-

tion either of the housekeeping gene glyceraldehyde phosphate de-

hydrogenase (GAPDH) or of one of the following specific genes:

1(II) collagen, osteocalcin, and lipoprotein lipase (LPL). The

PCR reactions were performed in a 100-L mixture that contained

1 PCR buffer, 2 mM MgCl2, 1 mM each of dCTP, dATP, dGTP,

and dTTP, 30 pmol of 5 and 3 specific primers, and 2.5 U of Am-

pliTaq DNA polymerase. Primer sequences for all the aforemen-

tioned genes were as follows: GAPDH: 5-ACCACAGTCCAT-

GCCATCAC-3 and 5-TCCACCACCCTGTTGCTGTA-3 [29];

1(II) collagen: 5-CTGCTCGTCGCCGCTGTCCTT-3 and 5-

AAGGGTCCCAGGTTCTCCATC-3 [11]; OC: 5-CATGAG-

AGCCCTCACA-3 and 5-AGAGCGACACCCTAGAC-3 [30];

LPL: 5-GAGATTTCTCTGTATGGCACC-3 and 5-CTGCAA-

ATGAGACACTTTCTC-3 [30]. The expected product sizes were

GAPDH 450 bp, 1(isoform IIB) collagen 225 bp, OC 310 bp, and

LPL 276 bp. The number of cycles used was in the linear range of

amplification for the specific gene product, as preliminary experi-

ments were carried out by the authors originally describing the


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primer sets. The reaction profiles used were as follows, with all

preceded by a first denaturation step at 95

C for 4 min: GAPDH:

95C for 1 minute, 55C for 1 minute, and 72C for 1 minute, for

24 cycles; OC: 95C for 1 minute, 55C for 1 minute, and 72C for

1 minute, for 30 cycles; LPL: 95C for 1 minute, 60C for 1

minute, and 72C for 1 minute, for 35 cycles; 1(II) collagen:95C for 3 minutes, 58C for 2 minutes, and 72C for 1.5 minutes,

for one cycle and then 95C for 1 minute, 58C for 1 minute, and

72C for 1.5 minute, for 30 cycles. All PCR reactions ended with a

7-minute incubation at 72C. RT-PCR products were analyzed by

electrophoresis of 20-L aliquots in 1% agarose gels and visual-

ized by ethidium bromide, except for the 1(II) fragment obtained

by the BMSC RNAs that were ethanol precipitated to load the en-

tirety of the products. The intensity of individual bands was quan-

tified by image analysis using the public domain software NIH

1.60. The amount of PCR product for each single gene was nor-

malized according to the corresponding GADPH PCR product.

Results

Growth kinetics and maintenance

of differentiation potential of primary BMSC cultures

The growth kinetics of three different BMSC cultures was

investigated from the primary culture through the fifth pas-

sage, corresponding to about 24 doublings. Expanded

BMSC were tested for their ability to undergo osteogenic,

chondrogenic, and adipogenic differentiation after appropri-

ate induction until the fourth passage.

Primary cultures reached the first confluence in about 3

weeks and 1215 doubling (Fig. 1). On replating, BMSC

slowed their proliferation rate considerably and by the sec-

ond passage stabilized their growth rate at about 1014 daysper population doubling (Fig. 1 and Table 1). The average

doubling time increased from 1.3 days at the primary cul-

ture stage to 7.7 at the first passage and 14.7 at the third pas-

sage (Table 1). Concurrent with this slowing of growth rate,

BMSC showed a change in appearance, from the initial

spindle shape to a more flattened square morphology (data

not shown).

Differentiation in the three lineages was induced as de-

scribed in the Materials and methods section. Figure 2

shows the results of a representative experiment with

BMSC at their first confluence. As revealed by immu-

nostaining with specific antibodies, OC expression was evi-

dent after the second week in osteogenic culture medium

(Fig. 2A), type II collagen was observed earlier, after 1

week in chondrogenic medium (Fig. 2C), and adipogenic

differentiation was always the last to be observed. Although

sporadic adipocytes already were evident after several days

in adipogenic medium, only after 3 weeks of stimulation did

cultures display several large adipocytic colonies, as re-

Figure 1. BMSC culture growth kinetics and differentiation potentialmaintenance. Growth kinetics of three BMSC cultures was determined.

Primary cultures reached the first confluence in about 3 weeks and 1215

doublings. On replating, BMSC slowed their proliferation rate consider-

ably. The curves represent the cumulative doubling number of the three

individual cultures versus time in culture.

Table 1.

Growth kinetics and differentiation of one BMSC primary culture

Differentiation

Passage Total doublings Average doubling time (days) Osteogenesis Chondrogenesis Adipogenesis

0 14.6 1.3

1 17.2 7.7

2 18.7 10.0

3 20.2 14.7

4 22.2 9.5

5 24.1 15.8 ND ND ND

Population doublings and differentiation potential of one BMSC culture were determined at the first confluence (passage 0) and at each passage thereafter.

Average doubling time at each passage was determined by dividing the number of doublings undergone by the culture since the previous passage by the time

(in days) taken to reach confluence. Differentiation was assayed by staining with alizarin S (osteogenesis), alcian blue (chondrogenesis), and Sudan black (ad-

ipogenesis). Scoring was as follows: positive; borderline; negative, as defined in the Materials and methods section. ND not done.


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vealed by phase contrast microscopy and Sudan black stain-

ing (Fig. 2E). von Kossa, alizarin S, and alcian blue staining

were positive on similar cultures maintained in osteogenic

and chodrogenic medium, respectively (data not shown),

Control unstimulated cultures yielded barely detectable lev-

els of expression of the differentiation markers (Fig. 2B, D,

and F).Differentiation in the three lineages induced by culture

with inductive media was verified by semiquantitative RT-

PCR analysis using primers for type II collagen, OC, and

LPL (Fig. 3 and Table 2). As shown in Figure 3 (lane 5), un-

stimulated cultures already show some, although variable,

basal expression of the specific makers studied:

1(II)

(panel B) and LPL (panel C) were clearly detectable,

whereas the OC level (panel A) was barely above the back-

ground. With appropriate stimulation (Fig. 3, lane 6), the

expression of the three markers was significantly induced.

With respect to the basal level, the OC,

1(II), LPL levels

were increased by a factor of 16, 3.8 and 4.8, respectively(Table 2). In long-term unstimulated cultures, the levels of

1(II) and LPL were similar to or lower than the basal level,

showing absence of spontaneous differentiation (Table 2

and Fig. 3 B and C lane 7). In contrast, the level of OC

expression in long-term unstimulated cultures was about

three times higher than the basal level (Table 2 and Fig. 3A,

lane 7).

For the three markers considered, the expected sizes of

the PCR products were consistent with those obtained with

Figure 2. Differentiation of human BMSC. Confluent human BMSC cul-

tures were maintained in differentiation medium (A, C, E) and in standard

control medium (B, D, F) for different time intervals. Osteocalcin deposi-

tion in the extracellular matrix, as revealed by immunostaining, was posi-

tive after the second week in osteogenic medium (A). After 1 week in

chondrogenic medium, discrete matrix noduli were positive for type II col-

lagen (C). Several large adipocytic colonies were observed with Sudan

black staining after 3 weeks of stimulation (E). Without the stimulating

factors, cultures kept in the same conditions for the same time period dis-

played only basal levels of differentiation (B, D, F). Immunostaining for

osteocalcin (A, B), immunostaining for type II collagen (C, D), and Sudan

black staining (E, F). Bar 1 mm.

Figure 3. RT-PCR analysis for expression of osteoblast, chondrocyte, and

adipocyte-related genes in BMSC, bone, cartilage, and adipose tissue.

RNA was extracted from unstimulated proliferating BMSC ( lanes 2 and 5),

from confluent cells stimulated for the individual lineage (lanes 3 and 6),

and from parallel cultures of confluent unstimulated cells (lanes 4 and 7).

RNA also was extracted from native tissues as control for PCR products

(lanes 8 and 9): bone (A), cartilage (B), and adipose tissue (C). Reverse

transcribed cDNA was amplified by using specific oligonucleotide primers

(described in the Materials and methods section). PCR products were visu-

alized in ethidium bromide stained gels. Lanes 2, 3, 4, and 8 correspond to

GADPH product, lanes 5, 6, 7, and 9 (A) to OC product, lanes 5, 6, 7, and 9

(B) to 1(II) product, and lanes 5, 6, 7, and 9 (C) to LPL product. A 123-bp

ladder was used as standard (lane 1: from top to bottom 492, 369, 246,

123 bp).

Table 2. Quantification of PCR products in differentiating

BMSC cultures

Condition OC 1(II) LPL

Basal expression 1 1 1

Induced differentiation 16 3.8 4.8

Spontaneous differentiation 2.9 0.7 0.6

RT-PCR reaction products shown in Figure 2 were quantified by image

analysis. For each set of reactions, product intensity was corrected accord-

ing to the amount of the corresponding GAPDH PCR product and normal-

ized to the relative basal level.


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RNA extracted from the corresponding native tissues (Fig.

3AC, lane 9).

Parallel with the loss of proliferation velocity was a loss

of differentiation potential (Table 1). By the 19th doubling,

BMSC greatly reduced their response to adipogenic stimu-

lation to a borderline level and completely lost it by the

22nd passage. Chondrogenesis, as revealed by alcian blue

staining, and osteogenesis, as assessed by alizarin S stain-

ing, were maintained longer, but dropped to borderline liv-

els by the fourth passage, corresponding to about 22 dou-

blings.

In vivo bone-forming efficiency

Osteogenic potential of freshly harvested bone marrow

cells, which include both osteogenic and hemopoietic cells,

was investigated and compared to that of expanded primary

BMSC, which reached confluence in about three weeks, un-

dergoing 17.5 population doublings. Samples of fresh bone

marrow were adsorbed onto HA ceramic cubes and subcuta-

neously implanted in nude mice as described in the Materi-als and methods section. We first determined that when 5

million nucleated cells from fresh bone marrow after sepa-

ration on Ficoll were implanted, the amount of bone tissue

formed was roughly comparable to that observed in the ex-

periments where the standard 250,000 expanded BMSC

were used. Hemopoietic marrow was detected in both speci-

mens (data not shown).

Several independent experiments were performed, and

the data from one representative experiment are given in

Table 3. It should be noted that over the 3-week culture pe-

riod, only about 14% of the expanded BMSC remained clo-

nogenic (data not shown). When the capacity to promotebone formation in the nude mice assay was evaluated, we

observed that the osteogenic potential of these expanded

CFU-F decreased about 36 times with respect to CFU-F in

fresh bone marrow. We found that, with fresh marrow, 1

mm3 of bone could be formed for every 161 CFU-F im-

planted, whereas, with expanded BMSC, 1 mm3 could be

produced for every 5,781 CFU-F implanted, on average.

Lifespan and maintenance

of differentiation potential of BMSC clones

One hundred twenty BMSC clones were obtained from five

different marrow samples, of which 80 were able to reach

confluence after passing from the cloning 96 well into one

well of a 24-well plate. At this stage, they were divided into

four replicas for induction of differentiation. Only three of

the seven theoretically possible phenotypes (O, C, A, OC,

OA, CA, OCA) were observed: 34% of the clones were os-

teo-chondro-adipogenic (tripontential or OCA clones), 61%

were osteo-chondrogenic (bipotential or OC clones), and

5% were purely osteogenic (O clones).

Lifespan and the ability to maintain differentiation po-

tential with time in culture were determined for three OCA

and two OC clones (Table 4). OCA clones were able to

reach 2223 doublings after about 80 days of culture (Fig.4). All had lost adipogenic potential when tested at passage

3 (equivalent to 22 doublings), but maintained their osteo-

chondrogenic potential. The two OC clones had a slightly

shorter lifespan, reaching 19 and 22 doublings, respectively.

At passage 3, one clone lost its chondrogenic poten-

tial, whereas the other clone maintained its bipotentiality

(Table 4).

Discussion

The data presented in this study indicate that, after in vitro

culture, the population of expanded BMSC progressively

lost their ability to proliferate, differentiate in multiple mes-

enchymal lineages, and efficiency in generating mature

bone tissue. This effect is clearly evident after the first pas-

Table 3. Osteogenic efficiency of fresh bone marrow versus primary

expanded BMSC

Fresh marrow* Expanded BMSC

Population doublings 0 17.5

Total bone formed (mm3)

by 103 CFU-F 6.19 0.17

Osteogenic unit 161 5,781

In vivo implants were performed with 2.5 105 expanded BMSC or 5

106 nucleated cells from fresh marrow and the CFU-F content of the cul-

tures was determined as described in the Materials and methods section.

From the results of these experiments, the expected yield of 103 fresh or ex-

panded CFU-F and the sizes of the respective osteogenic units were calcu-

lated.

* Average of triplicate samples of one representative experiment. Average of duplicate samples of one representative experiment. Number of clonogenic CFU-F necessary to induce the formation of 1

mm3 of bone when implanted as described in the Materials and methods

sections.

Table 4. Lifespan and differentiation potential of tripotential and

bipotential clones

Clone

Doubling

no. O C A

Doubling

no. O C A

1 19 22

2 19 23

3 19 23

4 18 19

5 19 22

Clone lifespan ranged between 19 and 23 cell doublings. OCA clones

reached 2223 doublings, whereas OC clones had a slightly shorter

lifespan, reaching 19 and 22 doublings. The three OCA clones lost adipo-

genic potential by 22 doublings, although they maintained osteo-chondro-

genic potential. Of the two OC clones tested, one lost its chondrogenic poten-

tial, whereas the other clone maintained both osteogenic and chondrogenic

potential.

positive; negative.

A adipogenic phenotype; C chondrogenic phenotype; O osteo-

genic phenotype.


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sage and is possibly already occurring during the primary

culture, as indicated by the bone-forming efficiency assay.

This behavior is consistent with a population undergoing

commitment and differentiation, although this appears to be

a gradual process.

The presence in the BMSC population of progenitors for

both the mesenchymal family of tissues and for hemopoi-

esis-regulating stroma, together with the fact that these cells

can be easily isolated from bone marrow aspirates and can

be conveniently expanded and transfected in vitro, has

prompted a wave of interest in their exploitation as vehicles

for cell and gene therapy of both hematologic and musculo-skeletal pathologies.

In the various therapeutic settings currently under inves-

tigation, the need for BMSC to display stem cell features

varies considerable. When used for hemophilia B gene ther-

apy, BMSC are required to engraft and survive in a site with

access to the general circulation and secrete factor IX with-

out necessarily proliferating and differentiating; therefore,

the presence of stem cells is not the main issue. On the other

hand, if the reinfused BMSC are to reconstitute the he-

mopoiesis-supporting stroma or, even more so, are to sus-

tain healthy osteogenesis for the whole life of the recipient,

possibly a young child, clearly what will determine the last-

ing success of the procedure is the number of stem cells or

very early progenitors present in the reinfused population.

We sought, therefore, to study BMSC stem characteristics

during culture expansion.

Concepts currently accepted as defining a stem cell are

the self-renewing capability, the high capacity for cell divi-

sion (lifespan) maintained throughout the lifetime of the or-

ganism, and the multipotential differentiation capability

[31]. These criteria might not apply to cells of postnatal

mesenchymal tissues, where the apparent cell plasticity

might play the same role without the need for a real stem

cell compartment, given the low tissue turnover in adult-

hood [4,9]. In other words, the existence of the MSC in the

postnatal organism is still debatable.

To test the self-renewing ability of expanding BMSC, we

investigated the growth kinetics of primary cultures and

monitored their ability to differentiate into the three lin-

eages considered at each passage.Gene expression for lineage-specific markers was ana-

lyzed by RT-PCR and confirmed the immunochemical data.

Interestingly, low but detectable levels of expression for the

three markers considered were present before stimulation,

indicating a possible commitment of some the these cells to

differentiate into the three lineages tested already at the pri-

mary culture stage. Noteworthy is that, in long-term unstim-

ulated cultures, only the level of osteocalcin expression was

increased spontaneously with respect to the basal value.

This may indicate that the osteogenic pathway is the pre-

ferred lineage through which these cells progress, possibly

because of an intrinsic commitment or because the in vitroculture conditions represent a microenvironment favoring

osteogenesis.

The kinetics of the differentiation to the three lineages

during mitotic expansion show that adipogenesis starts fad-

ing by 18 doublings and is totally lost by 22 passages, when

chondrogenesis and osteogenesis start weakening. In agree-

ment with Bruder et al. [32], we found a slowing of cell pro-

liferation as a function of increasing passages. Although

these authors report a faster proliferation rate at every pas-

sage considered, this may reflect different culture condi-

tions, whereas the trend toward decreasing replication poten-

tial with increasing cumulative doublings is in concordance

with our data.This orderly loss of differentiation potential, paralleling

that of proliferation velocity, strongly points to a progessive

commitment of the population as a whole and to the inabil-

ity, in these culture conditions, of the putative stem cells in

the expanding population to self-renew and maintain a

pluripotent differentiative capability.

Interestingly, the largest drop in proliferation rate in all

three primary cultures occurs on the first passage, whereas

the differentiation potential is maintained for one passage

longer. At the primary culture stage, CFU-F are plated to-

Figure 4. Lifespan and differentiation potential of BMSC tripotential

clones. The lifespan of three OCA clones was determined and the mainte-

nance of the original differentiation potential at increasing cell doublings

was verified. They reached 2223 doublings in about 80 days of culture.

By then, they had lost any apparent capability of proliferating. The threee

clones maintained the original differentiation potential up to 19 doublings,

but they lost the adipogenic potential by the 22nd doubling still retaining

the OC potential The curve represents the average cumulative doubling

number of the three clones versus time in culture ( SE). The differen-

tiation ability is represented by the underlying histograms. Arbitrary units:

0 negative; 1 borderline; 2 positive, as defined in the Material and

methods section.


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gether with the whole marrow microenvironment: a part of

this is made of nonadherent cells (mainly hemopoietic) and

is removed with the medium changes, but a part is surely

adherent to the plastic or the stromal cells themselves (mac-

rophages, endothelial cells, and early hemopoietic progeni-

tors at least) and can be responsible for a complex network

of soluble and cell-contact signals to the BMSC. It is tempt-

ing to speculate that dilution of this nonproliferating com-

partment with the first passage can be related to BMSC loss

of proliferation and multidifferentiative ability. Further ex-

periments are needed to test this hypothesis.

It is possible that stem cells are present in the initial cul-

ture and that they undergo unequal division during expan-

sion, producing a vast progeny of committed and differenti-

ating daughter cells and becoming progressively diluted

during the following passages. The nearest approximation

to the putative MSC that can be studied is the CFU-F.

Therefore, we compared the osteogenic efficiency of native

CFU-F in fresh marrow to that of expanded CFU-F in first

confluence BMSC. The result was that first confluence ex-panded CFU-F were 36 times less efficient than fresh mar-

row samples at generating bone. The in vivo bone formation

assay depends on the presence in the sample of a sufficient

number of putative stem cells and/or early progenitors able

to proliferate and give rise to a consistent differentiated

progeny. Therefore, after in vitro expansion of about 17

doublings, which is in the range necessary to obtain a thera-

peutically useful numbers of cells, the clonogenic popula-

tion in a BMSC primary culture is significantly depleted of

osteogenic early progenitors/putative stem cells in compari-

son with a fresh bone marrow sample.

Results of experiments we performed on a number ofclones to determine their actual lifespan are consistent with

the results obtained from primary cultures. Results indicate

that these cells are able to undergo a limited number of mi-

totic divisions, with characteristic kinetics, progressing

faster at the primary culture level, gradually slowing down

during the following passages, and reaching a complete stop

after 2223 cell doublings. Futhermore, the three OCA

clones analyzed displayed a loss of adipogenic potential by

doubling 22, and one of two OC clones lost its chondro-

genic potential by 22 cell doublings.

Ex vivo expansion is necessary to obtain a sufficient

number of cells for in vitro engineering and use in novel

strategies of gene and cell therapy. When the reinfusedBMSC are used to sustain healthy histogenesis for the rest

of the life of the patients, possibly young children, as in the

case of osteogenesis imperfecta, the stem cell content of the

population delivered clearly is of paramount importance

and the effects of the in vitro expansion stage must be con-

sidered. However, it is not known how many stem cells/

early progenitors are needed in the reinfused population to

obtain a therapeutic benefit, and it is possible that a limited

expansion would be compatible with a successful proce-

dure. A better understanding of the culture conditions re-

sponsible for BMSC maintenance in an earlier progenitor

state is desirable.

AcknowledgmentsPartially supported by grants from Associazione Italiana Ricerca

sul Cancro, Italian Space Agency (ASI) and European Space

Agency (ESA). We wish to thank the Bone Marrow TransplantCentres at both S. Martino Hospital and G. Gaslini Pediatric Hos-

pital for their factive collaboration and Prof. G. Corte for helpful

discussion.

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Banfi 2000 Experimental-Hematology