No increased trapping of multipotentmesenchymal stromal cells in bone marrow filters

compared with other bone marrow cells

M Sundin1, M Remberger1,2, H Lonnies1, B Sundberg1, O Ringden1,2 and

K Le Blanc1,3

1Division of Clinical Immunology, Department of Laboratory Medicine, Karolinska Institutet, and 2Centre for Allogeneic Stem Cell

Transplantation, and 3Hematology Centre, Karolinska University Hospital Huddinge, Stockholm, Sweden

Background

Multipotent mesenchymal stromal cells (MSC) are candidates for

cellular therapy in regenerative medicine and as treatment of graft-

versus-host-disease (GvHD) after hematopoietic stem cell (HSC)

transplantation. It has been suggested that MSC may be trapped in

bone marrow (BM) filters during the stem cell procurement and lost

from the HSC graft.

Methods

We investigated filtered BM and filters from six HSC donors. MSC

were expanded from the two sources and investigated by flow cytometry,

doubling capacity, differentiation ability and suppression in mixed

lymphocyte cultures.

Results

A range of 0.3�3.4% cells was trapped in the filters. By flow cytometry,

there was no difference in the proportions of different cell types between

the filter-retrieved and filtered BM cells. The phenotype, immuno-

suppressive capacity, differentiation and growth were equal in MSC

expanded from the two cell sources.

Discussion

Given the low number of trapped cells, filters do not appear to be a good

source of MSC. When intended for clinical transplantation, MSC need

to be expanded ex vivo to achieve sufficient doses for a clinical effect.

Keywords

graft-versus-host disease, immunosuppression, stem cell.

IntroductionMultipotent mesenchymal stromal cells (MSC) [1], pre-

viously referred to as mesenchymal stem cells, have been

used to improve the outcome of hematopoietic stem cell

transplantation (HSCT) [2,3]. Reversion of therapy-resis-

tant grade IV acute graft-versus-host-disease (GvHD) of

the skin, gastrointestinal tract and liver and prolonged

survival have been seen after treatment with MSC [4,5].

The cells may have a wider role in regenerative medicine

and treatment of autoimmune disorders because of in vitro

proliferative capacity, ability to differentiate into more

mature lineages and immunosuppressive features [6�12].MSC may be regarded as ‘universal donors’ because they

seem to avoid rejection and reduce alloreactivity, despite

expression of HLA class I and even when induced to

express HLA class II [2,8].

In HSCT the cell dose is known to play an important

role and it has been shown that bone marrow (BM) grafts

rich in cells are associated with a better outcome [13�19].It is unclear whether this results from hematopoietic or

non-hematopoietic cells in the graft. Non-hematopoietic

cells, which support long-term hematopoiesis, commonly

form complexes with hematopoietic cells. These com-

plexes are trapped in marrow filters during the procure-

ment [20,21]. Vicente et al. [22] detached cells trapped in

BM filters and infused them into HSCT recipients

along with the transplant, which resulted in reduced

acute GvHD and transplantation-related mortality. It was

Correspondence to: Dr Mikael Sundin, MD, Division of Clinical Immunology, F79, Karolinska Institutet, Karolinska University Hospital

Huddinge, SE-141 86 Stockholm, Sweden. E-mail: mikael.sundin@ki.se.

Cytotherapy (2008) Vol. 10, No. 3, 238�242

– 2008 ISCT DOI: 10.1080/14653240801965164

postulated that the retrieved cells became an additional

dose of MSC, resulting in reduced acute GvHD and

enhanced cell counts post-transplantation.

In the present study, we investigated whether the cells

trapped in BM filters display MSC features and therefore

could explain the above findings. Cells were cultured,

induced to differentiate, assayed by flow cytometry and

used in mixed lymphocyte cultures (MLC).

MethodsBM and filter cells

In the operating room, BM was harvested from six healthy

donors for clinical purposes. Donors had given informed

consent and our study was approved by the regional ethics

review board. Aspirated BM was injected into transfusion

bags through a 200-mm filter made of a co-polymer

(Green line B83; CODAN Medizinische Gerate GmbH &

Co. KG, Lensahn, Germany). An aliquot of the filtered

BM was withdrawn for this study and the rest was used in

clinical transplantation. BM filters were collected and

filled with 2 mM EDTA (Invitrogen, Paisley, UK) diluted

in phosphate-buffered saline (PBS) at 88C. Thereafter thefilters were shaken, the fluid aspirated and the filters

refilled three times. After this procedure the filters

appeared clean compared with the yellowish color before.

Filtered BM and filter-retrieved cells were counted in

Turk staining.

MSC isolation and culture

MSC were isolated and cultured from filtered BM and

cells retrieved from filters as previously described in detail

[8]. Briefly, mononuclear cells were separated and cultured

in medium supplemented with 10% fetal calf serum (FCS;

Invitrogen). The serum lot was selected on the basis of

optimal MSC growth, with maximal retention of osteo-

genic, chondrogenic and adipogenic differentiation. The

cell doubling time was calculated using the following

equation: doubling time�t/(log2 (y/x)), where t�time in

culture, y�cell count at confluence and x�cell count at

start.

Flow cytometric characterization of

filtered marrow cells, filter-retrieved cells

and isolated MSC

Filtered BM cells, filter-retrieved cells and isolated

third-passage MSC were assayed by flow cytometry.

The cells were incubated with monoclonal antibodies

against CD3, CD14, CD34, CD45 and CD73 (BD

Biosciences, San Jose, CA, USA), CD29 (Coulter, Miami,

FL, USA), CD105 (DakoCytomation, Carpentaria, CA,

USA) and CD166 (Serotec, Oxford, UK). Non-specific

fluorescence was determined using equal aliquots of cell

preparation incubated with isotype controls (BD Bios-

ciences). Finally, the cells were assayed in a flow

cytometer and analyzed with Cellquest software (BD

Biosciences). Fluorescence signals from 10 000 cells were

counted and recorded.

Differentiation ability of MSC isolated from

filtered BM and filter-retrieved cells

The ability of second-passage MSC, isolated from BM and

filter-retrieved cells, to differentiate into the more mature

cells, i.e. osteogenic and adipogenic cell types, was

investigated as described previously [10]. The MSC

differentiated into osteogenic and adipogenic cells were

stained with alizarin red S staining and oil red O solution

(Sigma-Aldrich, St Louis, MO, USA), respectively, and

morphologically examined by microscopy. Thereafter, the

staining were dissolved in 10% cetylpryidium chloride and

100% isopropanol, and assayed at 562 nm and 500 nm in a

spectrophotometer, respectively.

Inhibition of lymphocyte proliferation by MSC

isolated from filtered BM and filter-retrieved

cells

Peripheral blood lymphocytes (PBL) were isolated from

volunteer donors and used in MLC, to determine the

ability to suppress lymphocyte proliferation by addition of

MSC from filtered BM and filter-retrieved cells. Isolation

of PBL and MLC was performed as described elsewhere

[23,24].

Statistics

All comparisons between the cells, filtered BM or filter-

retrieved, and MLC with and without MSC, were tested

using a t-test in Statistica software (Statsoft Inc., Tulsa, OK,

USA).

Results and discussionSmall amounts of cells were trapped in

the BM filters

A mean of 0.09�109 (range 0.02�0.182) nucleated cells

was retrieved from the BM filters, which averaged 1.1%

(range 0.3�3.4) of the total amount of aspirated nucleated

No increased trapping of MSC in filters 239

cells. The filter-retrieved cells were 68% (median, range

56�82) mononucleated. Two of the pediatric donors

exhibited the highest cell doses retrieved, whereas the

other donors showed comparable results (Table 1). It

was evident that some BM cells were trapped in the

transfusion-set filters during the procurement. More cells

seemed to be retrieved from filters of pediatric donors,

which may be because pediatric BM is richer in cells than

adult BM.

Equal distribution of cell types in filtered

BM and filter-retrieved cells

The cells were analyzed in three regions according to size

and thereafter expression of the above-mentioned surface

markers. A high expression of hematopoietic markers

(CD3, CD14, CD34 and CD45) was shown in comparable

levels for filtered BM and filter-retrieved cells. In both

groups, cells negative for hematopoietic markers were

found in equal amounts. Cells negative for hematopoietic

markers but positive for unspecific MSC markers (CD73,

CD105 and CD166) could be demonstrated at equal and

small numbers in both filtered BM cells and cells retrieved

from filters (data not shown). The recent report by Vicente

et al. [22] suggested that MSC were trapped in BM filters

to a greater extent compared with other BM cells. Our

study could not confirm that MSC were more prone to be

trapped, as the expression of surface markers was compar-

able regardless of the source. MSC size is 30 mm versus

filter pores of 200 mm, so MSC should pass through filters

when they are not in complex formations. However, the

MSC may bind the highly polar plastic in our filters

because the cells are adhesive and consist of proteins

(Professor M. Hedenqvist, personal communication). To

minimize the potential loss of cells, which could be

beneficial in the case of ‘poor marrow’ [14], filters of less

polar materials should be used.

No differences in MSC phenotype

Characterized by flow cytometry, culture-expanded MSC

isolated from filtered BM and filters uniformly expressed

CD73, CD90 and CD105. The cells did not express the

hematopoietic markers CD3, CD14, CD34 and CD45. No

differences between the two groups could be demonstrated

(Figure 1A).

No statistically significant differences in the doubling

time during MSC culture could be demonstrated between

MSC isolated from filtered BM and filter-retrieved cells.

The doubling time was c. 4 and c. 6 days for both groups in

first and second passages, respectively. In the third passage,

the doubling time was c. 4 and c. 8 days for MSC isolated

from filter-retrieved cells and filtered BM, respectively

(data not shown). Doubling time increasing over time is

commonly seen.

The ability of the MSC from the two sources to

differentiate was equal (data not shown). In the absorbance

analysis of osteogenic and adipogenic differentiation, cells

isolated from BM filters exhibited values of 4.6 and 4.4,

compared with 4.3 and 3.3, respectively, for cells isolated

from filtered marrow (p�0.05). MSC from both sources

was immunosuppressive in vitro, as addition to MLC

significantly suppressed lymphocyte proliferation (Figure

1B).

The effect of reduced GvHD after infusion of filter-

retrieved cells, as reported by Vicente et al. [22], may be

explained by trapping of large doses or particularly

immunosuppressive MSC. Our results indicate that MSC

are not trapped to a greater extent than other cells in BM

filters. Therefore, a higher incidence of GvHD in

transplantation of filtered BM cannot be explained by

Table 1. Details of donors and cells

MSC 82 84 86 89 91 93

Donor (sex, male or female/age, in years) M/1 F/19 M/7 M/45 F/55 M/4

Number of filters 1 3 1 4 5 1

Total cell dose aspirated �109 5.1 18.7 7.4 12.6 19 6.8

Total cell dose retrieved �109 0.172 0.182 0.02 0.035 0.084 0.061

Percentage cells trapped in filter 3.4 1 0.3 0.3 0.4 0.9

Mononucleated cell dose retrieved�109 0.1 0.5 0.01 0.02 0.06 0.05

Percentage mononucleated cells retrieved 71 72 57 56 74 82

240 M Sundin et al.

loss of MSC. We have also demonstrated that MSC

isolated from filtered BM and filter-retrieved cells do

not differ in phenotype (expression of surface markers,

doubling time, differentiation capacity). Immunosuppres-

sion in MLC was equally potent using the MSC isolated

from the two sources, indicating that the trapped MSC do

not posses enhanced MSC features.

BM filters are not a good source of MSC

As MSC are present in low numbers in human BM and as

only small amounts of cells are trapped in BM filters, it

appears unlikely that MSC are responsible for the

beneficial effect of giving filter-retrieved cells to HSCT

recipients. To completely rule out possible MSC involve-

ment, unique surface markers that allow prospective

isolation of MSC are needed. However, to reduce GvHD

in HSCT larger numbers of cells seem to be needed and

therefore clinical ex vivo expansion of MSC, from BM

aspirations, should be applied [3,5].

AcknowledgementsThis study was supported by unrestricted grants from the

Swedish Cancer Society (Katarina Le Blanc, 4562-B05-

05XCC; Olle Ringden, 0070-B06-20XBC), the Children’s

Cancer Foundation (Katarina Le Blanc, 05/007; Olle

Ringden, 06/094), the Swedish Research Council (Katarina

Le Blanc, K2006-32X-14716-04-1, K2005-32P-15457-01A;

Olle Ringden, K2007-64X-05971-27-1), the Tobias Foun-

dation (Katarina Le Blanc), the Cancer Society in Stock-

holm (Katarina Le Blanc and Olle Ringden), the Swedish

Society of Medicine (Katarina Le Blanc), the Stockholm

County Council (Katarina Le Blanc), the Sven and Ebba-

Christina Hagbergs Foundation (Katarina Le Blanc),

the Cancer and Allergy Foundation (Olle Ringden), the

Signe och Olof Wallenius Foundation (Mikael Sundin), the

Foundation Sigurd och Elsa Goljes Minne (Mikael

Sundin), the Blodcancerfonden (Mikael Sundin) and the

Claes Hogman’s SAGMAN-scholarship/Fenwal Blood

Technologies Inc. (Mikael Sundin) and Karolinska

Institutet (Mikael Sundin, Katarina Le Blanc and Olle

Ringden).

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