Isolation of mesenchymal stem cells from G-CSF-mobilized human ...

Bone Marrow Transplantation (2006) 37, 967–976 & 2006 Nature Publishing Group All rights reserved 0268-3369/06 $30.00

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ORIGINAL ARTICLE

Isolation of mesenchymal stem cells from G-CSF-mobilized human peripheral blood using fibrin microbeads I Kassis1, L Zangi1, R Rivkin1, L Levdansky1, S Samuel2, G Marx3 and R Gorodetsky1,3 1 Radiobiology and Biotechnology Laboratory, Sharett Institute of Oncology, Hadassah-Hebrew University Medical Center, Jerusalem, Israel; 2Department of Bone Marrow Transplantation, Hadassah-Hebrew University Medical Center, Jerusalem, Israel and 3HAPTO Biotech Ltd, Jerusalem, Israel

Adult mesenchymal stem cells (MSC) that are able to differentiate into various mesenchymal cell types are typically isolated from bone marrow, but their significant presence in human peripheral blood (PB) is controversial. Fibrin microbeads (FMB) that bind matrix-dependent cells were used to isolate MSC from the mononuclear fraction of mobilized PB of adult healthy human donors treated with a granulocyte colony-stimulating factor. Isolation by plastic adherence resulted in a negligible number of MSC in all samples tested, whereas FMBbased isolation yielded spindle-shaped cell samples that could further expand on plastic or on FMB in eight out of the 11 samples. The yield of these cells at days 17–18 after the harvest was B0.5% of the initial cell number. The isolated cells were grown on plastic and characterized by FACS analysis and immunohistochemistry for specific markers. Following culturing and first passage, the FMBisolated cells stained positive for mesenchymal stromal cell markers CD90 and CD105, expressed vimentin and fibronectin and were negative for hematopoietic markers CD45 and CD34. These cells could differentiate into osteoblasts, adipocytes and chondrocytes. This study indicates that FMB may have special advantage in isolating MSC from sources such as mobilized PB, where the number of such cells is scarce. Bone Marrow Transplantation (2006) 37, 967–976. doi:10.1038/sj.bmt.1705358 Keywords: mesenchymal stem cells (MSC); granulocyte colony-stimulating factor (G-CSF); mobilization; fibrin microbeads (FMB); differentiation; human peripheral blood

Correspondence: Dr R Gorodetsky, Radiobiology and Biotechnology Laboratory, Sharett Institute of Oncology, Hadassah-Hebrew University Medical Center, POB 12000, Jerusalem 91120, Israel. E-mail: rafi@hadassah.org.il Received 28 November 2005; revised and accepted 27 February 2006

Introduction The presence of non-hematopoietic stem cells in the bone marrow (BM) was first suggested by Fridenshtein.1,2 These cells were shown to be able to differentiate into a number of mesenchymal phenotypes, including osteocytic and chondrocytic cells. Subsequently, these cells were isolated and defined as mesenchymal stem cells (MSC). They consisted of fibroblast-like adherent cells with high proliferative capacity.3–5 Other compartments in different tissues were also proposed as possible sources for MSC, including adipose tissue,6,7 liver,8 first-trimester blood8 and fetal pancreas.9 The ability to separate a significant number of adult MSC from other sources, such as cord blood (CB) and peripheral blood (PB), is controversial.10–14 The isolation of a high number of potent MSC sets the basis for new methods for tissue regeneration and cell therapy. Nevertheless, the procedure of BM extraction is traumatic and the amount of material extracted is limited. Therefore, exploring new sources and isolation techniques for obtaining such cells is of great interest. Stem cell mobilization is a procedure that induces rapid proliferation of hematopoietic stem cells in the BM and release of high number of immature stem cells into the bloodstream.15 The presence of a small number of MSC was detected in mobilized PB of healthy patients as well as those with malignancies.13,16–23 Most of the studies suggested that some MSC may also be present in nonmobilized PB in healthy donors or in patients with malignant diseases, but they are too few to be detected and cultured for a long term.13,16,22 Fibrinogen, the major component of the blood coagulation process, also seems to play a crucial role in tissue repair by providing an interim matrix that supports cell adhesion, proliferation and migration involved in wound repair.23,24 Based on these observations, three-dimensional cultures based on fibrin matrices were proposed for cell culture for cellular tissue repair therapy.23–33 Fibrin microbeads (FMB) were proposed as a particulate form of fibrin more suited to cell culture conditions. Essentially, the FMB were formed from fibrin in a heated oil emulsion system that permitted crosslinking by endogenous factor XIII. The heat was kept relatively low (below 801C) to retain the cell attachment epitopes of

FMB for MSC isolation from mobilized blood I Kassis et al

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native fibrinogen.25 Fibrin microbeads of 50–250 mm diameter were formed.25,27,29 Based on their high binding efficiency of matrix-dependent cells, the FMB were used to selectively isolate and expand MSC derived from mouse and rat BM. The isolated cells could be driven to differentiate on the slowly biodegradable FMB27,28 and delivered with the FMB that also serve as vehicles for carrying the cell into the target tissues.27 These qualities of the FMB make them the candidates of choice for various tissue engineering applications. In a preliminary study, we showed that FMB were able to separate MSC from a population of rat BM at a higher yield than the conventional isolation method based on plastic adherence.26,27 Furthermore, we showed that mesenchymal cells isolated by FMB from rat BM could form bone structures both in vitro and in vivo.27 These qualities make the FMB a choice matrix for various tissue engineering applications. In the present study, we evaluated the ability of FMB to separate human MSC from different sources other than BM, with special emphasis on granulocyte colony-stimulating factor (G-CSF)-mobilized PB of healthy individuals. We showed that FMB were able to isolate significant numbers of MSC from the mobilized PB progenitor cells (PBPC) of adult healthy individuals, with low contamination by other cell types. The cells that were isolated and expanded on FMB proved their pluripotency by their ability to differentiate into various phenotypes from the mesodermal lineage.

Materials and methods Fibrin microbeads preparation and use Fibrin microbeads were produced from concentrated fibrinogen by a stirred heated oil emulsion technique as previously described.26–29 The diameter of FMB used for this study was in the range of 105–180 mm. After production, the FMB were stored dried and sterilized by immersion in sterile 70% ethanol for at least 8 h before use, or by g-irradiation (15 kGy). The dried FMB have prolonged shelf life at room temperature (RT) without losing their activity. Before their use for cell separation, the FMB were washed and hydrated three times with sterile Dulbecco’s phosphate-buffered saline (PBS Biological Industries, Beit-Haemek, Israel). The hydrated FMB increased in size by about 30% but were resistant to degradation in ethanol, organic solvents and aqueous solutions of neutral pH for prolonged periods of at least a few weeks. Peripheral blood progenitor cell samples derived from mobilized peripheral blood The blood mobilization process was performed to prime the BM of normal donors. It consisted of two daily injections for 5 days 10 mg/kg G-CSF – Neupogens (Amgen Inc., Thousand Oaks, CA, USA). Following the G-CSF treatment, the mononuclear polymorphonuclear cells were separated and collected from the fraction rich in mononuclear cells of the PB of the donor, using a cell separator (COBE Spectra Apheresis System operated with version 6.1 Bone Marrow Transplantation

PBSC software, Gambro BCT, Lakewood, CO, USA). Basically, anticoagulant heparin solution was added to the blood to keep it from clotting during the procedure and the blood/anticoagulant mix cycled through a centrifuge to separate the mononuclear and stem cells from the other components and plasma of the PB. The separated cell system pumped the separated cells into a collection bag, whereas the other blood components and plasma were returned to the donor. The collection of cells was carried out at the interface of 1–2% hematopoietic cell transplantation. The main final output was composed of 70–90% mononuclear and B10–15% polymorphonuclear cells, which were used for transfusion to donors. The samples for our experiments were obtained from minute residual waste materials remaining after such PBbased BMT procedure. The use of this source of material was approved by Hadassah Helsinki Ethics Committee as a use of waste samples from human sources for research purposes, with no tracking of the donor’s identity and with no DNA examinations or manipulations.

Cell culture media The main media used were either ‘poor’ or ‘rich media’ (PM and RM, respectively). Poor media were based on Dulbecco’s modified Eagle’s medium with low glucose (1.0 g/ml) þ 10% fetal calf serum (FCS) þ 1% vitamins þ 1% non-essential amino acids þ 1% glutamine þ 1% Pen/ Strep (all from Biological Industries, Bet-Haemek, Israel). Rich media consisted of Dulbecco’s modified Eagle’s medium with high glucose (4.5 g/ml) þ 20% FCS þ 1% vitamins þ 1% non-essential amino acids þ 1% glutamine þ 1% Pen/Strep (all from Biological Industries, Bet-Haemek, Israel). Preparation of peripheral blood progenitor cells for isolation of mesenchymal stem cells The PBPC samples were washed ( 3) with PBS by centrifugation for 6 min at 1100 r.p.m. (241C). Cell debris was removed and the samples of PBPC were re-suspended with fresh medium. The cells were then subjected to the two MSC isolation methods, by plastic adherence or by FMB, as described below. Mesenchymal stem cells isolation by adherence to plastic Peripheral blood progenitor cells were seeded on plastic plates in ‘rich’ or ‘poor’ medium supplemented with 20 U/ml heparin (Sigma-Aldrich, Rehovot, Israel). Cultures were maintained at 371C in a humidified atmosphere containing 7% CO2 for 48 h, then the plates were washed with PBS to remove non-adhered cells and the medium was exchanged. The cultures were maintained for an additional 18–19 days with twice weekly medium exchange. Fibrin microbeads-based mesenchymal stem cells isolation method For the isolation of MSC by FMB, a total number of B100 106 PBPC were mixed with B100 ml packed volume of hydrated FMB in 50 ml tubes with perforated cap to allow gas exchange. The perforated cap was


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wrapped loosely with a sheet of aluminum foil to allow gas exchange, as previously described.25 Poor or rich medium was supplemented with 20 U/ml heparin. The tubes were placed in an angle of B201 in a slowly rotating device placed in a cell culture incubator at 371C with 7% CO2 for 48 h.27 For collection of cells loaded on the FMB, the medium was removed after 48 h of incubation with the nonattached cells and the FMB were washed with PBS. The washed cells loaded on FMB were mixed with 2–3 mg/ml diluted fibrinogen (Vitex, New York, NY, USA, or Omrix, Tel Aviv, Israel, or equivalent source). The FMB were dispersed on plastic dishes and extra fibrinogen was pumped out. Thrombin (10 U/ml; Vitex, New York, NY, USA, or Omrix, Tel Aviv, Israel) was then sprayed on the plate to form a thin fibrin film that immobilized the FMB loaded with cells to the plastic surface by polymerizing the minute residual fibrinogen in the plate. Medium (RM or PM) was then carefully added and the FMB attached to the plastic dishes were incubated for an additional 18–19 days. Cultures were maintained at 371C in a humidified atmosphere containing 7% CO2 with medium exchange twice weekly.

Cell nuclei staining To indicate the density of cells loaded on FMB, their nuclei were stained with propidium iodide (PI) (Sigma-Aldrich, Rehovot, Israel). A few microliters of suspended FMB were fixed with 500 ml ethanol (70%) for 10 min at RT, then 5 ml of PI (5 mg/ml) added for 7 min in darkness. The stained FMB þ cells were then mounted on a microscope slide with mounting solution. The red-stained nuclei of cells attached to FMB were visualized by fluorescence microscopy. Flow cytometry for cell antigens Cells cultured on plastics were trypsinized and fixed with fresh paraformaldehyde (4%) for 20 min at RT. For staining of intracellular components, the cells were permeabilized by incubation with 0.5% Triton100 (Sigma-Aldrich, Rehovot, Israel) for 7 min in RT. For blocking non-specific binding, cells were rinsed with 5% bovine serum albumin (BSA) in PBS for 60 min at RT on a slowly plane-rotating plate. The cell suspension was then incubated with the following primary antibodies to the different markers : mouse anti-human CD45 (1:10), mouse anti-human CD34 (1:10), mouse anti-human CD90 (1:10), mouse anti-human CD105 (1:10), all from Serotec, Oxford, UK, mouse anti-human fibronectin (1:500) and rabbit antihuman vimentin (1:50) both from Sigma-Aldrich, Rehovot, Israel. The antibodies were diluted to required concentrations with FACS buffer containing 1% BSA þ 0.01% sodium azide (Sigma-Aldrich, Rehovot, Israel) in PBS. For their assay, the cells were incubated with the following secondary antibodies: donkey anti-mouse-FITC-conjugated (1:100) or donkey anti-rabbit-FITC-conjugated (1:100) both from Jackson Labs, Maine, USA, diluted in FACS buffer on a slowly plane-rotated plate for 45 min at RT in darkness. Rinsing–washing of the cells between different steps was carried out by short microfuge centrifugation. Cells were suspended in 400 ml FACS buffer and then filtered through a mesh sieve of 50–90 mm into

5 ml FACS tubes. From each sample, 10 000 cells were analyzed with FACScan (BD Biosciences, Franklin Lakes, NJ, USA).

Immunostaining Adhered cells were trypsinized for 7–10 min with 0.25% trypsin/EDTA (Biological Industries, Bet-Haemek, Israel), washed and resuspended in 24-well plates (B5 104 cells/ well). Cells were incubated at 371C, 7% CO2 for 24 h. Medium was discarded and cells were washed gently with 0.05% Tween 20 diluted in PBS ( 3). Cells were fixed with fresh paraformaldehyde (PFA, 4%) for 20 min at RT. For staining of intracellular components, cells were permeabilized with 0.5% Triton100 (Sigma-Aldrich, Rehovot, Israel) added for 7 min. For blocking unspecific binding, cells were rinsed with 5% BSA (Biological Industries, BetHaemek, Israel) in PBS for 60 min at RT on a slowly planerotated plate. Then, cells were washed by 0.05% Tween 20 diluted in PBS ( 3) and were incubated with the following primary antibodies: mouse anti-human CD45 (1:10), mouse anti-human CD34 (1:10), mouse anti-human CD90 (1:10), mouse anti-human CD105 (1:10), mouse anti-human fibronectin (1:500) and rabbit anti-human vimentin (1:50) diluted to required concentrations with FACS buffer containing 1% BSA. Cells were washed gently ( 3) in PBS with 0.05% Tween 20. For detection, cells were incubated with the following secondary antibodies: goat anti-mouse-Cy3-conjugated (1:100), goat anti-rabbit-Cy2-conjugated (1:100) both from Jackson Immunoresearch Laboratories, West Grove, PA, USA, diluted in 1% BSA buffer, on a slowly plane-rotated plate for 45 min in darkness at RT. Cells were mounted with mounting solution and viewed by fluorescence and light by Nomarsky’s optics microscopy. Assays for mesenchymal stem cells differentiation For osteogenic differentiation, PM was used with 105 M dexamethasone, 50 mg/ml ascorbic acid and 10 mM bglycerophosphate (Sigma, Israel). Confluent MSC were cultured with differentiation factors for 21 days with two medium exchanges per week. Adipogenic differentiation was induced using MesenCultt basal medium (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with adipose stimulatory supplements: 1-methyl-3-isobutylxanthine, 109 M dexamethasone, 5 mM insulin and 5 mM indomethacin (Stem Cell Technologies, Vancouver, BC, Canada). Confluent MSC were cultured with differentiation factors for 21 days with three weekly medium exchange. Chondrogenic differentiation was induced by culturing the cells with PM containing 10 ng/ml TGF-b3 (Sigma-Aldrich, Rehovot, Israel). Confluent MSC were cultured with differentiation factors for 14 days with medium exchange three times a week. Cytochemical analysis of the differentiated cells Alizarin red staining was used to stain mineralized matrices for the detection of bone-like structures formed by monolayer cell culture. Differentiated cells were fixed with a solution of 4% formaldehyde–methanol–water (in the ratio of 1:1:1.5), then stained with saturated solution of alizarin red (pH 4.0) (Sigma-Aldrich, Rehovot, Israel) for Bone Marrow Transplantation


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15 min at RT, washed with water and air dried. The surface areas covered with red stain represented mineralized nodules. Nitro-blue tetrazolium/indolylphosphate (NBT/ BCIP) staining was also used to detect expression of alkaline phosphatase, which indicated osteoblast activity. The NBT/BCIP staining was carried out with substrates of nitro-blue tetrazolium (NBT, non-colored) mixed with indolylphosphate (BCIP) yielding a blue color owing to the enzyme activity. Before staining, the cells were washed with PBS, 0.5 ml of NBT/BCIP (ICN Biomedicals, Irvine, CA, USA) was added and they were then incubated at 371C in a humidified atmosphere containing 6% CO2 for 30 min. The cells were washed with PBS and fixed with 4% PFA. The surface areas that stained blue represented alkaline phosphatase activity. For the detection of adipogenic differentiation, accumulated intracellular lipid droplets were detected by oil-red-O staining. Cells were washed ( 3) with PBS. A 0.25 ml portion of oil-red-O solution (10 mg/ml) (Sigma-Aldrich, Rehovot, Israel) was added to the cells for 20 min at RT. Cells were washed with PBS, and fixed with 4% PFA. Under the light microscope, round intracellular lipid drops were stained orange. For the detection of chondrogenic differentiation, alcian blue staining was used. It stained mucopolysaccharides or glucosaminoglycans as markers of secreted chondrocytes matrix. The cells were then washed with PBS ( 3). For fixation, fresh 4% PFA was added for 20 min at RT. The cells were rinsed with alcian blue solution, pH 2.5 (Sigma, Israel) for 30 min at RT. They were then washed in running tap water for 2 min and in distilled water for another 2 min. Extracellular matrix components secreted by chondrocytes were then stained blue.

The cells on FMB were downloaded to plastic dishes from FMB from the 2nd day with adhesion of the beads to the plastics by a dilute fibrin clot. The cells expanded on the plastics and covered its surface with fibroblast-like cells within the next 18–19 days. With time, colonies of cells from the FMB expanded to form eventually a monolayer on the plastic surface as shown in Figure 1b–d. Then, the cells were harvested and counted to evaluate their yield. For the further expansion of these cells, they were recultured in RM with regular passages when 80–90% confluence was reached (Figure 1e). Cells with fibroblast-like morphology that could be further expanded were obtained by the FMB isolation method in eight of 11 PBPC samples examined. The ratio of the FMB-isolated cells after 18–19 days in culture relative to the total number of PBPC in the initial sample from which the MSC were isolated ranged between B0.3570.077% in PM and B0.5070.16% in RM. On the other hand, the number of surviving and proliferating cells by the conventional plastic adherence method in both culturing media was too low to allow a significant calculation of a yield and in none, a culture of ‘MSC-like’ cells could be established. In the plastic adhesion isolation technique, the PBPC that remained on the plate after 2 days of incubation did not reveal the fibroblast-like shape as seen with the FMB isolation method. Rather, most cells were larger and rounder and resembled leukocytes (Figure 1f). Following 18 days of incubation, most of these cells died in culture, yielding a negligible number of cells with mesenchymal phenotypes that could not be further expanded in culture.

Photomicrographs The samples were mounted on glass with mounting solution (Sigma, Israel). Pictures from microscopy were taken with a Nikon Coolpix camera with a Nikon Eclipse TE200 microscope with Nomarsky optics and fluorescence set-up. The pictures were taken with objective 10, 20, 40 and a zoom set-up to yield a full field width of 1, 0.5 and 0.25 mm, respectively. Fluorescence pictures were taken by manual setting (typically at an exposure time of 0.1–1 s, aperture of 6.4). In some cases, low-light exposure was superimposed on the fluorescence image to show the outlines of the structures.

Immunophenotypic characterization of peripheral blood progenitor cell-derived mesenchymal stem cells FACS analysis (Figure 2a and b) and immunohistochemistry (Figure 2c) were performed in parallel to identify the types of cells isolated by different methods. The cell markers used were as follows: CD45, a common leukocyte marker; CD34, hematopoietic stem cell marker; CD105 and CD90, vimentin and fibronectin as putative markers for MSC. Initially, before the isolation procedure, the vast population of PBPC was found to stain positive for CD45 and negative for the mesenchymal antigens CD90 and CD105 (Figure 2a). However, after the isolation procedure with FMB, harvesting (passage 0) and after 2–3 passages, the residual cells were typically found to be negative for hematopoietic CD45 and CD34, indicating the nonhematopoietic origin of these cells, whereas the positive staining for CD90, CD105, vimentin and fibronectin (Figure 2b) indicated their similarity to MSC derived from BM. It is noteworthy that the degree of hematopoietic cell contamination (detected by CD45 þ antigen) of the isolated cells by FMB adhesion after their harvest at 18–19 days (passage 0) may vary. In some samples, initially they reached up to B20% of the total cells (not shown). However, this residual fraction of CD45 þ cells was no longer present after 2–3 passages, as shown by FACS analysis (Figure 2b) and by immunohistochemistry (Figure 2c), whereas the residual adhered cells were recorded as being both CD90 and CD105 positive. In a

Results Isolation of mesenchymal stem cells using fibrin microbeads The FMB-based method for isolating MSC was based on adding hydrated FMB to the suspension of PBPC for 2 days in slow rotation in culture conditions in either PM or RM. Then, the FMB with attached cells were allowed to settle and the non-adhered cells in the suspension were evacuated. The FMB-attached cells were downloaded and further grown on plastic dishes in PM or RM for an additional 18–19 days. The density of cells attached to the FMB after 2 days could be visualized by fluorescence microscopy with PI nuclei staining (Figure 1a). Bone Marrow Transplantation


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Figure 1

Isolation of human mesenchymal stem cells (MSC) on fibrin microbeads (FMB) and their downloading on plastic dishes. (a) Fluorescence micrograph of cells loaded on FMB after 48 h of incubation with peripheral blood progenitor cells from mobilized blood in suspension. The FMB have faint self-fluorescence. The nuclei of cells bound to FMB are visualized by intense propidium iodide staining of the cell nuclei. (b–d) Downloading cells from FMB (with the aid of low concentrations of fibrin) after 7, 14 and 18 days in culture, respectively. (e) Near-confluent MSC population isolated with FMB following two passages on plastic dishes. (f) Typical phenotype of the cell population isolated by the conventional plastic adhesion method after 18 days of culture. These cells were easily detached and their survival was reduced so that after several passages they were eliminated.

few instances, cells isolated by the conventional plastic adherence method could be collected to allow FACS analysis before they died or detached. Most of these cells were found to be positive for CD45 and negative for CD90 and CD105 (not shown).

In vitro differentiation of osteocytes, adipocytes and chondrocytes from peripheral blood progenitor cell-derived mesenchymal stem cells To investigate the differentiation potential of the cells isolated by FMB to mesenchymal phenotypes, cells from Bone Marrow Transplantation


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a

CD45

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CD90

CD105

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CD45

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CD34

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CD90

80

Counts x 100

40

0 100

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CD105

101

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104 100

Fibronectin

101

102

103

104

Vimentin

80

40

0 100

101

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101

102 FL1

103 104 100

101

102

103 104

c CD45

CD34

Vim

FN

CD105

CD90

100 m Figure 2

Immunophenotype of mesenchymal stem cells isolated by fibrin microbeads (FMB). (a) FACS analysis of the immunophenotype profile for hematopoietic marker CD45 and for mesenchymal antigens CD90 and CD105 of the initial peripheral blood progenitor cell sample before isolation. Cells isolated by FMB technique were harvested 18–19 days following harvesting and cultured for further passages. Shaded histograms represent the fluorescence from control cells stained with only second antibody; non-filled histograms represent positive staining of cells with the indicated antibody. (b) Cells from passages 2–3 were harvested and labeled with antibodies against CD45, CD34, CD90, CD105, fibronectin (FN) and vimentin (Vim) and analyzed by FACS. Shaded histograms represent the fluorescence from control cells stained with only second antibody; non-filled histograms represent positive staining of cells with the indicated antibody. (c) Cells from passage 5 were harvested and immunostained with antibodies to CD45 (Cy3 labeled), CD34 (Cy3 labeled), CD90 (Cy3 labeled), CD105 (Cy3 labeled), FN (Cy3 labeled) and Vim (Cy2 labeled). The fluorescence images were merged with dim-light images of the same field to show cell contours.

early passage (passages 3–4) were cultured under conditions appropriate for inducing differentiation to osteogenic, adipogenic and chondrogenic lineages. The adipogenic potential was evaluated morphologically, where spindleBone Marrow Transplantation

shaped fibroblast-like cells were transformed to rounded cells that contained lipid vesicles, as visualized by the oilred-O staining (Figure 3a). Chondrogenic differentiation was detected by positive staining of alcian blue, which


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stains mucopolysaccharides or glycosaminoglycans of foci of chondrogenic cells (Figure 3b). When induced to differentiate to osteoblasts, the cells formed mineralized structures, as shown by alizarin red staining of calcium deposits (Figure 3d). Nitro-blue tetrazolium/indolylphosphate staining also showed high activity of alkaline phosphatase, typical of the activity of osteoblasts in the foci of differentiated cells (Figure 3c).

Differentiation of mesenchymal stem cells to bone-forming cells on fibrin microbeads To test the ability of the isolated FMB-loaded cells to differentiate into bone-forming cells, the MSC isolated from PBPC were cultured in suspension on the beads for 5 days in RM. Then, they were supplemented with bone differentiation medium for further 10 days. Nitro-blue tetrazolium/indolylphosphate staining was used to observe alkaline phosphatase activity of the osteogenic cells on the

a

FMB. Propidium iodide staining for nuclei showed the density of all the cells on the FMB as a reference. A blue staining was observed on most of the FMB loaded with cells, indicating osteogenic alkaline phosphatase activity and differentiation into osteogenic cells on most cells that loaded FMB (Figure 4b, black arrow). On some beads, all cells were stained, whereas in others, only part of them differentiated. This hints at the possibility that once a few cells initiate differentiation, they induce similar differentiation in neighboring cells that reside on the same bead (Figure 4a, white arrow). On plastic two-dimensional culture, cell differentiation was also evident in the formation of foci of high number of differentiated cells.

Discussion Major efforts have been made to examine techniques for isolation of MSC from PB with methods adapted from the

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Figure 3 In vitro differentiation of mesenchymal stem cells isolated by fibrin microbeads from mobilized peripheral blood. Mesenchymal stem cells, expanded by 3–4 passages, were driven to differentiate into (a) adipocytes, as visualized by oil-red-O staining that stains cellular oil droplets, and (b) chondrocytes that were detected by alcian blue staining, which stains matrix secreted by these cells. Differentiation to osteoblasts was demonstrated by the alkaline phosphatase activity detected by nitro-blue tetrazolium/indolylphosphate (c) and by secreted calcified matrices that were stained positive by alizarin red (d).

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a

Reports on isolation circulating human MSC/stromal cells

Study (ref)

Blood fraction

MSC/stromal cells isolated

Fernandez et al.16

PB, PBPC

Lazarus et al.21

PBPC

Zvaifler et al.22

Elutriated PB

|

Kuznetsov et al.17

PB

|

Wexler et al.13

PBPC

PB | PBPC

200 m

b

200 m

Notes Samples collected from breast cancer patients after chemotherapy Samples collected from cancer patients undergoing autologous infusion of PBPC Samples from healthy nonmobilized blood Initial volume of each blood sample was 50 ml and 7–8 steps of eluatriation was needed to reach a yield of 0.05% Only two colonies of adherent fibroblast-like cells were observed The isolated cells were CD45, CD14 and CD34 negative; however, they did not express mesenchymal markers such as CD105 and Stro-1 In vivo bone formation by the isolated cells was observed Cultures from PBPC showed a minimal sparse fibroblast-like cells that could not be passaged, and expressed CD45+ and CD14+

Figure 4

Abbreviations: MSC ¼ mesenchymal stem cells; PB ¼ peripheral blood; PBPC ¼ peripheral blood progenitor cell. | MSC/stromal–mesenchymal cells detected. MSC/stromal–mesenchymal cells not detected.

techniques for their isolation from BM.5 However, the success of this process was controversial.13,16–23 Some groups looked for MSC in samples of cord blood, in normal PB of adult donors or in G-CSF-mobilized PBPC in the PB of adult volunteers,10–13,16,22 as summarized in Table 1. But the results so far were controversial. Fibrin microbeads are not identical to native fibrin gels. They are composed of highly crosslinked, dense, stable and partially denatured three-dimensional fibrin matrix. The FMB do not exhibit procoagulant activity but retain the high cell-binding properties of native fibrin toward nonhematopoietic cells, including mesenchymal cells.29–32 The mechanism of cells binding to this fibrin-based matrix 24–25 may be at least partially attributed to the exposure of haptides, new cell-binding epitopes on fibrinogen that selectively adhere to matrix-dependent cells.34 Fibrin microbeads could be used for the isolation of MSC, for their expansion and differentiation. We previously reported that FMB could separate MSC from a suspension of mixed cell types with high efficiency.27 Other cell types may also

transiently attach to the FMB. Macrophages that may bind to FMB are not tightly bound and are detached within the isolation period of 48 h. Circulating endothelial cells may also attach FMB, but in the process of expansion, a negative selection leaves on the FMB only cells with fibroblastic phenotype. In previous reports, we showed that FMB-mediated MSC isolation from rat BM was 3–5 times higher in comparison to the conventional isolation technique of adherence to plastic.26 The current study shows the ability of FMB to isolate MSC from G-CSF of mobilized blood of healthy donors collected by apheresis. This procedure separates efficiently from the donor’s mobilized blood circulation a higher number of cells from the mononuclear fraction, which contain mostly hematopoietic stem cells for transplantation with relatively low contamination by other cell types. Mobilization may also increase the release into circulation of other BM-derived stem cells, including MSC, which may also be found in the mononuclear fraction. In eight out of 11 samples of mobilized blood, we isolated a significant number of MSC, that could be further expanded. The yield of isolated MSC from mobilized PB downloaded and expanded on the flasks at days 17–18 by

Differentiation of bone-like structures on fibrin microbeads (FMB). (a) Cells were loaded and proliferated on FMB for a prolonged period of about 2 weeks with osteogenic differentiation medium. The cell density on the FMB is shown by propidium iodide red fluorescence of cell nuclei (white arrow). (b) Staining with nitro-blue tetrazolium/indolylphosphate showing alkaline phosphatase activity of the cells that were driven to differentiate into bone-forming cells (black arrow).



the FMB technique was only B0.5%. This yield was lower than that we have previously reported for whole rat BM.26 Nevertheless, this small cell number was still sufficient to allow separation and expansion of such cells from most of the samples. By contrast, the number of cells isolated by conventional plastic adherence that could be further expanded was negligible in all the samples tested, as previously reported by other groups using the plastic separation methods.13,21,22 The fact that not all samples of mobilized blood could yield MSC, even with our FMB technique, may be attributed to different factors. It may be associated with the variability in the number of progenitors secreted upon G-CSF treatment. It may also be associated with the age and health condition of the donor and the exact time of aphaeresis after G-CSF administration. These factors should be further explored in order to improve the chances of recruiting MSC from mobilized blood. FACS analysis showed that most cells in the fresh PBPC samples were of hematopoietic origin, with positive expression for CD45 and negative expression for CD90 and CD105 (Figure 2a). Cells isolated by the conventional plastic adherence method yielded mostly non-fibroblastlike cells resembling the morphology of monocytes and macrophages (Figure 1f). Rare fibroblast-like cells were observed on the plastic plates, and they did not expand further. Culturing in different media (RM or PM) had no significant effect on the outcome. However, cells isolated by FMB and downloaded onto plastic were mostly spindle-shaped, with fibroblast-like morphology (Figure 1a–e). In some cases, these cells were initially contaminated with small population of round cells and monocytes. The best yield with highest cell purity was achieved when the cell isolation by FMB was performed in RM. The majority of cells isolated by FMB and further expanded in culture for 2–3 passages stained poorly for CD45 and CD34, which served as hematopoietic cell markers, and stained positive for mesenchymal and stromal markers CD105, CD90, vimentin and fibronectin (Figure 2b). The moderate expression of CD34 þ in the fraction of the FMB-isolated fibroblast-like cells in some of the samples is of interest (not shown), as BM-derived MSC were previously described as CD34 negative.5 Similar observations related to progenitor cells in PB of cancer patients were previously reported.16 This cell fraction may represent fibrocytes in mobilized blood that display adherent, spindle-shaped morphology when cultured in vitro and still retain leukocytic properties.35,36 These cells were reported to be able to migrate into sites of tissue injury and possibly play a role in the inflammatory process.35,36 Sub-populations of these cells were reported to be able to secrete collagen and fibronectin and still be positive for the hematopoietic markers CD45, CD13 and CD34.37,38 The variation in the proportion of the CD34 þ CD45 þ cells within the FMB-isolated cell populations may derive from difference in the number of fibrocytes in the bloodstream in different donors, although the mesenchymal differentiation of such cells as reported here should not be ruled out. The FMB-isolated MSC, which were downloaded onto plastic, could be further expanded. After 4–5 passages, the cell population appeared homogeneous. No significant

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contamination with rounded non-mesenchymal cells could be observed (Figure 2c). In order to get a clearer characterization of the differentiation potential of the isolated cells, they were driven to differentiate into different mesodermal cell types, which included osteocytes, chondrocytes and adipocytes (Figure 3). Indeed, the isolated progenitors showed indications for their mesenchymal nature, both morphologically and immunophenotypically. Mesenchymal stem cells isolated from human mobilized PB were able to expand and differentiate into bone-forming cells while still attached to the FMB (Figure 4). The implications of these findings are that the cells on the FMB can be driven to differentiate into the desired phenotype and then to be injected into a target organ while still on the biodegradable FMB. This may have major applications for regenerative medicine. For example, FMB could be used to separate and expand MSC from autologous mobilized blood obtained from the pheresis of mobilized PB. Following MSC expansion and differentiation into the desired phenotype, the same FMB loaded with cells could then be used to transplant these autologous cells into the target organ, without the need to trypsinize the cells from the FMB, which serve as their supporting biodegradable matrix. This may permit simpler delivery of the cells in conditions that allow for better survival and better chances to regenerate the wounded tissue.

Acknowledgements We thank Anna Hotovely-Solomon, Irena Shimeliovich and Elena Gaberman for their technical help and advice. This work was partially supported by the Israel Science Foundation Grant #697/001 to RG and by HAPTO Biotech Ltd.

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