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Could cord blood be a source of mesenchymal stromal cells for clinical use?
Cytotherapy (2008) Vol. 10, No. 5, 452459
Could cord blood be a source of mesenchymal stromal cells for clinical use? C Perdikogianni, H Dimitriou, E Stiakaki, G Martimianaki and M Kalmanti Department of Pediatric Hematology/Oncology, University Hospital of Heraklion, University of Crete Medical School, Heraklion, Crete, Greece
Background Cord blood (CB) has long been regarded as an easily accessible source of hematopoietic progenitors suitable for transplantation, but its efficiency as a source of mesenchymal stromal cells (MSC) remains controversial. The aim of this study was to assess CB as a potential source of MSC, to determine the optimal culture requirements for CB MSC expansion and to compare their functional and immunophenotypic characteristics with bone marrow (BM) MSC from children. Methods Mononuclear cells from 18 full-term CB samples and 23 BM samples from children were set in culture under MSC-inducing conditions. Their immunophenotypic characteristics were assessed by flow cytometry and their differentiation potential was evaluated.
serum (FCS) enrichment of the culture medium, high FGF-2 concentration and high sample volume. Isolated CB MSC were morphologically similar to the ones derived from BM, but appeared late in culture. An adherent cell layer was formed and reached confluency in 34 days (passage 1; P1) and needed 55 days subsequently (from P1 to P2). CB MSC retained their characteristics for two successive passages. Immunophenotypic analysis showed no expression of CD34 and varying expression of CD45, ranging from 0% to 17.83%, and CD105, from 49% to 83%. CFU-F colonies developed in one case. Discussion These findings suggest that CB cannot be considered a sufficient source of MSC for clinical use, although easily accessible. Further research should aim for alternative sources.
Results Isolation of CB MSC was achieved in 25% of the samples cultured under optimal conditions: high initial cell concentration, fetal calf
Keywords bone marrow, children, cord blood, mesenchymal stromal cells.
Introduction
tested the clinical use of these cells in the setting of local implantation in cases of tissue damage, for the treatment of autoimmune diseases such as rheumatoid arthritis, in tissue engineering protocols, in gene therapy and lately as tumortargeting drug delivery vehicles [36,9]. Wound healing has been shown to improve after MSC infusion and several animal models have dealt with repair of injured myocardium [4,6,10]. In pediatrics, mesenchymal cells have been used in some patients with osteogenesis imperfecta [9,11] as well as in a few patients with Hurler’s disease and metachromatic leykodystrophy several years after allogeneic hematopoietic stem cell transplantation [12]. Recent studies have demonstrated that MSC also have immunomodulatory effects exerted through different and still not fully elucidated mechanisms that include
Mesenchymal stem cells, now called mesenchymal stromal cells (MSC), constitute a rare population of multipotent progenitor cells with the capacity to differentiate into at least osteogenic, adipogenic and chondrogenic cell lineages and potentially into non-mesoderm type cells [13]. Their mesodermal origin and immunomodulating properties have led to assessment of their capacity to regenerate damaged tissues and support hematopoiesis. These features render them a promising candidate for cellular therapies [48]. Bone marrow (BM) mesenchymal cells can be readily isolated and differentiated in vitro under specific culture conditions. They account for 0.0010.1% of the whole population of nucleated cells [24,6,9]. Current trials have
Correspondence to: Professor M. Kalmanti, Department of Pediatric Hematology/Oncology, University Hospital of Heraklion, 71110 Heraklion, Crete, Greece. E-mail: [email protected]. – 2008 ISCT
DOI: 10.1080/14653240701883079
Cord blood mesenchymal stromal cells
suppression of T-cell activation and proliferation [9]. These properties make MSC a potent therapeutic tool against graft versus host disease (GvHD) in the transplantation setting [4,5,9] and they have already been given in patients with grade IIIIV GvHD with a substantial effect in a significant proportion of patients [5,9,13]. In addition, MSC have been studied for their ability to improve hematopoietic stem cell engraftment in vivo [9,14,15]. Friedenstein et al. [16] were the first to develop in vitro culture techniques for the isolation and differentiation of MSC, and further studies proposed the existence of a stromal stem cell to maintain the marrow microenvironment [17]. As the number and frequency of BM MSC decline with age, alternative MSC sources are required [18,19]. According to the literature, other potential sources of mesenchymal cells include the liver, fetal blood, cord blood (CB), amniotic fluid and adipose tissue [19,20]. Isolation and expansion of MSC from the subendothelial layer of the human umbilical cord vein [21,22] and placental tissue [23] have also been achieved. Not all investigators have accomplished isolation of MSC from human umbilical CB and thus data on this matter are controversial. Some investigators have reported that they could not isolate MSC from CB or mobilized adult peripheral blood [2426] whereas others have shown the ability to isolate MSC from pre-term CB [20,25,27]. The aim of this study was to isolate and characterize MSC from CB, seeking the optimal culture conditions that enhance their isolation and expansion and comparing them with BM MSC from children. Detecting differences between the two potential MSC sources (BM and CB) concerning culture conditions and functional and immunophenotypic characteristics of the isolated MSC could help our understanding of whether the properties of MSC are source-dependent.
hematologic disorders (idiopathic thrombocytopenic purpura, ITP, n 13; autoimmune neutropenia, n 10) were studied. The study was approved by the Ethical Committee of the University Hospital of Heraklion, Crete.
Isolation and expansion of CB MSC CB mononuclear cells (MNC) were isolated following FicollHypaque (d 1077 g/mL; Lymphoprep-Nycomed, Oslo, Norway) separation. Initially, the protocol for isolation and expansion of BM MSC was applied. The culture medium consisted of low-glucose Dulbecco’s modified Eagle medium (DMEM; Invitrogen Ltd, Paisley, UK), with 10% fetal calf serum (FCS; lot especially selected for optimal growth of MSC; Hyclone Perbio Science, Erembodegem AALST, Belgium), 200 mM Lglutamine, 10,000 U/mL penicillin and 10 mg/mL streptomycin (Invitrogen) and 1 ng/mL fibroblast growth factor-b (FGF-b; Abcys SA, Paris, France), at a cell concentration of 50 000 cells/cm2 in 25-cm2 T flasks (Corning, NY, USA). Different culture conditions were tried in order to optimize MSC isolation and expansion from CB, as shown in Table 1. The various conditions were assessed either alone or in combinations. Each modification of the standard protocol for MSC isolation from BM was initially tried alone and then consecutively in combination with each other until the optimal set of culture conditions was achieved. Pre-treatment of the culture surface with FCS was performed in order to avoid stable macrocytic adherence [29]. The culture surface was Table 1. Different culture conditions tried in order to optimize MSC isolation and expansion from CB Culture conditions
Range of conditions tried
Cell concentration, cells/cm2
4 105 1 106 3 106 10
Methods Samples CB samples from 18 full-term deliveries (16 normal labors and two Cesarean sections) were studied. The samples were collected after completion of labor using a standard protocol [28] in 50-mL vials containing 750 IU heparin (Sigma Chemicals, St Louis, MO, USA). The processing of all samples was performed within 24 h of collection. The samples did not contain blood clots nor showed signs of hemolysis, parameters that could affect their quality. In addition, 23 BM samples from children with benign
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FCS concentration in culture medium volume, % FGF-b concentration, ng/mL Culture surface, cm2 Sample volume, mL Pre-treatment of culture plate
(n 4) (n 13) (n 4) (n 5)
20 (n 13) 1 (n 6) 5 (n 11) 10 (n 9) 25 (n 9) B40 (n 8) 40 (n 10) With FCS (n 12) With fibronectin (n 2)
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also pre-treated with fibronectin (Sigma) for 45 min, at a concentration of 2 mg/cm2 of culture surface, in two experiments in order to enhance firm adhesion of the MSC on the culture flask. Non-adherent cells were removed within 24 h of initial plating, with a complete medium change performed once a week thereafter. When layers were confluent (70% confluency), cells were treated with 0.25% trypsin/1 mM EDTA (Invitrogen), counted and replated at a concentration that ranged from 1000 cells/cm2 (n 4) to 50 000 cells/cm2 (n 2) in 25-cm2 flasks for passage 1 (P1) and the passages thereafter.
Immunophenotypic analysis and assessment of functional characteristics of CB MSC The immunophenotypic analysis was performed according to the BM protocol. At day 0 of culture and at subsequent passages, 1 105 cells were stained for 20 min with antiCD45fluoroisothyocyanate (FITC), CD34phycoerythrin (PE), CD29FITC and CD105PE. Labeled cells were thoroughly washed with phosphate-buffered saline (PBS), with 2% FCS and sodium azide, and analyzed on an Epics-Coulter cytometer. For CFU-F assay, at day 0 of culture (the day of initial plating of MNC) 1 105 CB MNC in the standard culture medium described above (low-glucose DMEM with 10% FCS, 200 mM L-glutamine, 10 000 U/mL penicillin and 10 mg/mL streptomycin), without FGF-b, were seeded in each well of a 24-well plate (in triplicate). After 14 days of culture, CFU-F was quantified on an inverted microscope after staining with Giemsa.
Isolation and expansion of BM MSC All the BM samples were cultured under the following conditions. BM MNC, following FicollHypaque (d 1077 g/mL; Lymphoprep-Nycomed) separation, were cultured in a-MEM without nucleotides (Invitrogen), in the presence of 10% FCS (lot selected for optimal growth of MSC; Hyclone Perbio Science), 200 mM L-glutamine and 10,000 U/mL penicillin and10 mg/mL streptomycin (Invitrogen), at a cell concentration of 50 000 cells/cm2 in 25-cm2 T-flasks (Corning), with 1 ng/mL FGF-b (Abcys SA, Paris, France). A complete medium change was performed twice a week. When layers were confluent (85% stroma formation), cells were treated with 0.25% trypsin /1mM EDTA (Invitrogen) and replated at 1000
cells/cm2 (P1) in 25-cm2 T-flasks. MSC were maintained for a number of passages.
Immunophenotypic analysis and assessment of functional characteristics of BM MSC MNC were immunophenotypically characterized by flow cytometry analysis. At day 0 of culture and at various passages (P1P3), 1 105 cells were stained for 20 min with anti-CD45FITC, CD34PE, CD29FITC, CD90 FITC, CD105PE and CD146PE. Labeled cells were washed thoroughly with PBS, with 2% FCS and sodium azide, and analyzed on an Epics-Coulter cytometer. For a CFU-F assay, at day 0 of culture 1 105 BM MNC in the culture medium described above, without FGF-b, were seeded in each well of a 24-well plate (in triplicate). At subsequent passages MSC were plated in 20-cm2 Petri plates at a concentration of 10 cells/cm2 (in duplicate) and, following 14 days of culture, the CFU-F was quantified after staining with Giemsa. The cell doubling time (DT) was calculated using the formula: DT t log(2)/log(cells harvested/cells inoculated); t being the time between initial plating and harvest for the respective passage
Differentiation cultures At P2 or P3, the ability of BM MSC to differentiate into adipocytes (A), osteoblasts (O) and chondrocytes (C) was tested. Adipocyte differentiation MSC at P2 were plated in tissue culture flasks or plates at a cell density of 4 104 cells/cm2 and cultured in DMEM (Gibco BRL, Paisley, UK) supplemented with 20% FCS (Gibco), 100 nM roziglitazone (Cayman Chemicals, Ann Arbor, MI, USA), 1 mM dexamethasone, 60 mM indomethacin and 0.5 mM isobutylmethylxanthine (all from Sigma). The medium was replaced every 34 days for a total of 21 days. Osteocyte differentiation MSC at P2 were plated in tissue culture flasks or plates at a cell density of 1.5 104 cells/cm2 and cultured in DMEM supplemented with 10% FCS, 0.1 mM dexamethasone, 3 mM NaH2PO4 and 0.15 mM ascorbate-2-phosphate (Sigma). The medium was replaced every 34 days for a total of 21 days.
Cord blood mesenchymal stromal cells
Chondrocyte differentiation A micropellet culture system was applied in which 2.5 105 cells were centrifuged in a 15-mL polypropylene tube and the pellets cultured in DMEM supplemented with 0.1 mM dexamethasone, 1 mM sodium pyruvate, 0.17 mM ascorbate-2-phosphate, 0.35 mM proline (Sigma), 1 ITS premix (Cambrex Bio Science, Wokingham, UK), 5.35 mg/ mL linoleic acid (Sigma), 1.24 mg/mL bovine serum albumin (BSA) and 10 ng/mL TGF-b1 (R&D Systems, Abingdon, UK). The medium was replaced every 34 days for a total of 14 days. Differentiation was verified by histochemical staining (Oil red O staining for A, Von Kossa staining for O and Alcian blue staining for C) and reverse transciptasepolymerase chain reaction (RT-PCR) analysis (LPL expression for A, ALP and osteoprotegerin expression for O and collagen II expression for C). No differentiation experiments were performed for CB MSC due to the low number of cells harvested.
Results Isolation, expansion, immunophenotypic and functional characteristics of CB MSC Optimal culture conditions resulting in CB MSC isolation were found to be: (1) a higher initial concentration (3 106 cells/cm2) compared with both the BM protocol performed in our laboratory and other CB protocols already published; (2) enrichment of the culture medium with selected FCS (20% of the total volume compared with 10% of the BM protocol); (3) the addition of FGF-b at a higher concentration of 5 ng/mL compared with BM (1 ng/mL in the BM protocol); (4) pre-treatment of the culture surface with FCS; and (5) a high initial volume of the sample ( 40 mL). This set of parameters was characterized as optimal because of the achievement of MSC isolation only under the specific conditions combined together (n 8). Twenty-five per cent of the cultured CB samples formed an adherent layer of MSClike cells only under the optimal culture conditions. The isolated MSC were morphologically similar to the ones derived from BM but appeared later in culture because they needed 34 days from the initial MNC plating until confluency (n 2) and 55 days from P1 to P2 (n 1). The proliferative capacity of successfully isolated MSC, determined by the time needed from one passage to another, was low and they were not effectively passaged beyond the second passage, but retained their morphologic, adherent
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cell characteristics as long as they were in culture. CFU-F colony growth was evident in one case at P1. The total number of colonies obtained was 13. The immunophenotypic analysis of CB-derived MSC cells at P1 showed no expression of CD34 and variable expression of CD45 (017.83%), indicative of their hematopoietic origin. The CD105 expression exhibited a wide range of values (4983%), while another mesenchymal-related surface marker, CD29, was expressed at a range from 57.2% in one experiment to 88.5% in the other. No adequate number of MSC was obtained from CB in order to test the differentiation capacity. In every other case of modified culture conditions, apart from the optimal combination, either no adherent cells were isolated or a heterogeneous bi-morphic adherent cell population appeared (n 2). This population combined both the spindle-shaped and the round-shaped appearance (Figure 1), appeared 1316 days after initial plating of MNC and could be maintained in culture for up to 8 weeks, but without evidence of further expansion ability. In addition, it could not be successfully passaged and did not possess the immunophenotypic profile characteristic of MSC, as shown by the low expression of CD105 and high expression of hematopoietic markers.
Isolation, expansion, immunophenotypic and functional characteristics of BM MSC All BM samples studied successfully resulted in isolation and expansion of MSC. BM MNC formed an adherent stromal cell layer and reached confluency (P1) in approximately 1790.9 days. BM-derived MSC were adherent cells displaying the characteristic fibroblastic spindle shape. MNC at day 0 of culture expressed the hematopoietic markers CD34 and CD45, while there was low expression of mesenchymal-related markers CD105 (1.8590.3%), CD146 (0.9390.3%) and CD90 (2.69 1.1%). Expression of hematopoietic markers gradually decreased at subsequent passages, whereas expression of CD90 (96.591.47%), CD29 (96.692.7%), CD105 (99.49 0.23%) and CD146 (94.0591.45%) significantly increased. The mean number of CFU-F developed in culture was 42.995 at P1, while an average of 33.695 was obtained at P2. The proliferative capacity, as determined by the calculated cell DT, was 390.3 days at P1 and 4.390.6 days at P2. All BM MSC cultures successfully exhibited tri-lineage differentiation, as determined by histochemical analysis and further confirmed with molecular markers
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Figure 1. (a) Adherent cells from CB MNC in two experiments, with a heterogeneous bi-morphic phenotype that combined both the spindle-shaped and the round-shaped appearance. (b) BM-derived MSC at P2 with the characteristic spindle-shaped appearance.
assessed by RT-PCR (LPL expression for A, ALP and osteoprotegerin expression for O and collagen II expression for C). The comparison of phenotypic, functional and immunophenotypic characteristics of MSC derived from CB and BM is depicted in Table 2.
Discussion The ability of blood cells (peripheral blood or CB) to produce a stroma of mesenchymal cells seems to be restricted compared with the potential of BM MNC under similar culture conditions, possibly because of yet un-
defined culture requirements or the need for mobilization of circulating mesenchymal progenitors that have already migrated, together with hematopoietic cells, from the sites of early hematopoietic activity to BM at the time of labor [25]. In spite of reports on the ability of CB to generate MSC, their efficient isolation and expansion in adequate numbers for clinical use should still be considered controversial. MSC circulate in blood at a much lower concentration than that found in BM, specifically 0.052.8 non-hematopoietic progenitors/106 MNC plated in CB or 02.3 clones/1 108 MNC versus 25 MSC/106 MNC
Table 2. Comparison of phenotypic, functional and immunophenotypic characteristics of MSC derived from CB and BM
Morphology
Time to confluence, days CFU-F Immunophenotype, P1
Differentiation potential
CB
BM
Adherent cells with a spindle- shaped appearance similar to the BM MSC Two experiments with a bi-morphic adherent cell population, both spindle-shaped and round-shaped 3490 (n 2) 13 colonies at P1 (n 1)
Spindle-shaped appearance
CD34, 0% (n 2) CD45, 017.83% (n 2) CD105, 4983% (n 2) CD29, 57.288.5% (n 2) No adequate number of MSC obtained
1790.9 (n 23) 42.995 colonies at P1 (n 23) 33.695 colonies at P2 (n 23) 0% (n 23) 0% (n 23) 99.490.23% (n 23) 96.692.7% (n 23) In 23/23 samples Tri-lineage
Cord blood mesenchymal stromal cells
plated in BM [30,31]. This fact could contribute to the difficulty in isolating and expanding MSC from CB under the same culture conditions used for BM MSC. In the present study, CB MNC gave rise to an adherent cell layer with a mesenchymal-like phenotype and immunophenotypic characteristics similar to the relevant ones of BM-derived MSC at a percentage of 25%, when only the experiments with optimal conditions were taken into consideration. We initially tried to grow MSC from CB MNC using the technique already established in our laboratory for the isolation and proliferation of BM MSC. No expansion of adherent cells with MSC phenotypic characteristics was achieved and, therefore, different culture conditions were tried in order to enhance growth of MSC from CB. MNC were set in culture at a higher concentration than the ones used for BM MSC in the different protocols, the culture medium was enriched in FCS and the culture plates were pre-treated with FCS in order to prevent stable adherence of monocytic cells [29]. All the above parameters, in addition to a net sample volume of 40 mL or more and a high concentration of FGF-b in the culture medium, resulted in enhancement of the efficacy of CB MNC to generate a stroma of MSC in vitro. The scarcity of CFU-F in CB further supports the indications that stromal components are not present (or are present at very low frequencies) in CB. The phenotypic and immunophenotypic characteristics of CB-derived MSC were maintained for two subsequent passages, but the number of cells decreased at each passage and the time interval from the initiation of culture until confluency of the stroma was much longer compared with BM. These findings are in accordance with observations made by other scientists [26,30,32]. In some of our experiments, a heterogeneous bi-morphic adherent cell population that combined both the spindle-shaped and round-shaped appearance was observed, but this population could not be successfully passaged and possessed different immunophenotypic characteristics than MSC. One can speculate that other cells, such as macrophages, lymphocytes and other hematopoietic cells, could inhibit the growth of MSC [33]. Interestingly, according to the literature, even in the studies that report successful growth of MSC-like cells from mononuclear CB cells, MSC were not found in all the CB samples analyzed [2426]. The percentage of CB samples generating MSC, according to different reports, ranges from 23% to 63% [19,27,29,31,32], which contrasts
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with the ability of BM MNC to generate stroma of MSC in all the samples studied under the appropriate culture conditions. Variability in the number of MSC in individual CB units, also confirmed by other investigators [19,27,29,31,32], resulted in a wide range of percentage of successful MSC isolation. Erices et al. [27] and Campagnoli et al. [20] showed that mainly CB from preterm labors generated MSC, while another study by Yu et al. [25] showed that MSC mainly reside in fetal blood with a gestational age of 1626 weeks. In addition, Goodwin et al. [30] showed that MSC-like cells, termed non-hematopoietic progenitors, could be isolated from CB but needed more time to grow in culture compared with their BM counterparts. In agreement with this observation, Yang et al. [31] reported that more than half of the CB harvests that generated MSC-like cells were observed during the third week of the culture period. The investigators mentioned above reported a 23% success rate in isolating MSC, and only 2.2% of them with a high expansion capacity. Chang et al. [32] reported a slower appearance of CB MSC, by12 weeks, and a markedly reduced CFU-F frequency, by one-tenth, compared with BM. The differences observed concerning the success rate of isolating MSC from CB, compared with the 100% success rate for BM MSC, pose questions regarding the efficiency of using CB MSC in the clinical setting. Moreover, fewer CFU-F can be generated from CB MSC than from BM MSC, and different CFU-F assays compared with BM have been used by other investigators in order to induce CFU-F growth [30], otherwise impossible because of the low frequency of MSC in CB [33]. However, it has been reported that, although barely isolated, CB MSC showed the highest proliferative capacity compared with BM and adipose tissue but, strikingly, showed no adipogenic differentiation capacity [19]. It should also be mentioned that there is a report of successful isolation of CB MSC from CB MNC by negative immunodepletion of CD3 , CD14, CD19 , CD38, CD66b and glycophorin A cells and limiting dilution in order to obtain single-cell derived, clonally expanded, MSC [34]. Another work supports the isolation of CB MSC by CD133 selection, as the CD133 cell fraction contains more MSC with high proliferative potential [33]. In contrast, BM MSC from children with benign hematologic diseases, in the present study, proliferated in
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culture and were capable of generating an adherent stroma layer in approximately 2 weeks. Immunophenotypic analysis revealed a population of non-hematopoietic cells with a surface antigen profile characteristic of MSC. The immunophenotypic characteristics of MSC could be maintained throughout subsequent passages, as could their proliferative capacity. In addition, it was shown that BMderived MSC had a differentiation potential for adipocytes, chondrocytes and osteocytes, while no adequate number of MSC was obtained from CB in order to test their differentiation capacity, as has also been reported by other investigators [25]. According to the literature, although both BM and adipose tissue MSC demonstrate a multilineage differentiation capacity, there is a debate regarding whether CB MSC can be differentiated towards the adipogenic lineage [19,32]. In addition, Goodwin et al. [30] did not show an ability of chondrogenic differentiation in the CB MSC that they had isolated. Transplantation of MSC is currently under intense investigation and finding alternative sources of these cells, apart from BM, is mandatory because of the limitations that BM could pose. As effective cellular therapy requires a cell type that can be easily isolated and expanded, the findings of the present work suggest difficulties in considering CB an efficient source of MSC for clinical use, even though is easily accessible. Intense work should be concentrated on the search for alternative MSC sources.
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Could cord blood be a source of mesenchymal stromal cells for clinical use? C Perdikogianni, H Dimitriou, E Stiakaki, G Martimianaki and M Kalmanti Department of Pediatric Hematology/Oncology, University Hospital of Heraklion, University of Crete Medical School, Heraklion, Crete, Greece
Background Cord blood (CB) has long been regarded as an easily accessible source of hematopoietic progenitors suitable for transplantation, but its efficiency as a source of mesenchymal stromal cells (MSC) remains controversial. The aim of this study was to assess CB as a potential source of MSC, to determine the optimal culture requirements for CB MSC expansion and to compare their functional and immunophenotypic characteristics with bone marrow (BM) MSC from children. Methods Mononuclear cells from 18 full-term CB samples and 23 BM samples from children were set in culture under MSC-inducing conditions. Their immunophenotypic characteristics were assessed by flow cytometry and their differentiation potential was evaluated.
serum (FCS) enrichment of the culture medium, high FGF-2 concentration and high sample volume. Isolated CB MSC were morphologically similar to the ones derived from BM, but appeared late in culture. An adherent cell layer was formed and reached confluency in 34 days (passage 1; P1) and needed 55 days subsequently (from P1 to P2). CB MSC retained their characteristics for two successive passages. Immunophenotypic analysis showed no expression of CD34 and varying expression of CD45, ranging from 0% to 17.83%, and CD105, from 49% to 83%. CFU-F colonies developed in one case. Discussion These findings suggest that CB cannot be considered a sufficient source of MSC for clinical use, although easily accessible. Further research should aim for alternative sources.
Results Isolation of CB MSC was achieved in 25% of the samples cultured under optimal conditions: high initial cell concentration, fetal calf
Keywords bone marrow, children, cord blood, mesenchymal stromal cells.
Introduction
tested the clinical use of these cells in the setting of local implantation in cases of tissue damage, for the treatment of autoimmune diseases such as rheumatoid arthritis, in tissue engineering protocols, in gene therapy and lately as tumortargeting drug delivery vehicles [36,9]. Wound healing has been shown to improve after MSC infusion and several animal models have dealt with repair of injured myocardium [4,6,10]. In pediatrics, mesenchymal cells have been used in some patients with osteogenesis imperfecta [9,11] as well as in a few patients with Hurler’s disease and metachromatic leykodystrophy several years after allogeneic hematopoietic stem cell transplantation [12]. Recent studies have demonstrated that MSC also have immunomodulatory effects exerted through different and still not fully elucidated mechanisms that include
Mesenchymal stem cells, now called mesenchymal stromal cells (MSC), constitute a rare population of multipotent progenitor cells with the capacity to differentiate into at least osteogenic, adipogenic and chondrogenic cell lineages and potentially into non-mesoderm type cells [13]. Their mesodermal origin and immunomodulating properties have led to assessment of their capacity to regenerate damaged tissues and support hematopoiesis. These features render them a promising candidate for cellular therapies [48]. Bone marrow (BM) mesenchymal cells can be readily isolated and differentiated in vitro under specific culture conditions. They account for 0.0010.1% of the whole population of nucleated cells [24,6,9]. Current trials have
Correspondence to: Professor M. Kalmanti, Department of Pediatric Hematology/Oncology, University Hospital of Heraklion, 71110 Heraklion, Crete, Greece. E-mail: [email protected]. – 2008 ISCT
DOI: 10.1080/14653240701883079
Cord blood mesenchymal stromal cells
suppression of T-cell activation and proliferation [9]. These properties make MSC a potent therapeutic tool against graft versus host disease (GvHD) in the transplantation setting [4,5,9] and they have already been given in patients with grade IIIIV GvHD with a substantial effect in a significant proportion of patients [5,9,13]. In addition, MSC have been studied for their ability to improve hematopoietic stem cell engraftment in vivo [9,14,15]. Friedenstein et al. [16] were the first to develop in vitro culture techniques for the isolation and differentiation of MSC, and further studies proposed the existence of a stromal stem cell to maintain the marrow microenvironment [17]. As the number and frequency of BM MSC decline with age, alternative MSC sources are required [18,19]. According to the literature, other potential sources of mesenchymal cells include the liver, fetal blood, cord blood (CB), amniotic fluid and adipose tissue [19,20]. Isolation and expansion of MSC from the subendothelial layer of the human umbilical cord vein [21,22] and placental tissue [23] have also been achieved. Not all investigators have accomplished isolation of MSC from human umbilical CB and thus data on this matter are controversial. Some investigators have reported that they could not isolate MSC from CB or mobilized adult peripheral blood [2426] whereas others have shown the ability to isolate MSC from pre-term CB [20,25,27]. The aim of this study was to isolate and characterize MSC from CB, seeking the optimal culture conditions that enhance their isolation and expansion and comparing them with BM MSC from children. Detecting differences between the two potential MSC sources (BM and CB) concerning culture conditions and functional and immunophenotypic characteristics of the isolated MSC could help our understanding of whether the properties of MSC are source-dependent.
hematologic disorders (idiopathic thrombocytopenic purpura, ITP, n 13; autoimmune neutropenia, n 10) were studied. The study was approved by the Ethical Committee of the University Hospital of Heraklion, Crete.
Isolation and expansion of CB MSC CB mononuclear cells (MNC) were isolated following FicollHypaque (d 1077 g/mL; Lymphoprep-Nycomed, Oslo, Norway) separation. Initially, the protocol for isolation and expansion of BM MSC was applied. The culture medium consisted of low-glucose Dulbecco’s modified Eagle medium (DMEM; Invitrogen Ltd, Paisley, UK), with 10% fetal calf serum (FCS; lot especially selected for optimal growth of MSC; Hyclone Perbio Science, Erembodegem AALST, Belgium), 200 mM Lglutamine, 10,000 U/mL penicillin and 10 mg/mL streptomycin (Invitrogen) and 1 ng/mL fibroblast growth factor-b (FGF-b; Abcys SA, Paris, France), at a cell concentration of 50 000 cells/cm2 in 25-cm2 T flasks (Corning, NY, USA). Different culture conditions were tried in order to optimize MSC isolation and expansion from CB, as shown in Table 1. The various conditions were assessed either alone or in combinations. Each modification of the standard protocol for MSC isolation from BM was initially tried alone and then consecutively in combination with each other until the optimal set of culture conditions was achieved. Pre-treatment of the culture surface with FCS was performed in order to avoid stable macrocytic adherence [29]. The culture surface was Table 1. Different culture conditions tried in order to optimize MSC isolation and expansion from CB Culture conditions
Range of conditions tried
Cell concentration, cells/cm2
4 105 1 106 3 106 10
Methods Samples CB samples from 18 full-term deliveries (16 normal labors and two Cesarean sections) were studied. The samples were collected after completion of labor using a standard protocol [28] in 50-mL vials containing 750 IU heparin (Sigma Chemicals, St Louis, MO, USA). The processing of all samples was performed within 24 h of collection. The samples did not contain blood clots nor showed signs of hemolysis, parameters that could affect their quality. In addition, 23 BM samples from children with benign
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FCS concentration in culture medium volume, % FGF-b concentration, ng/mL Culture surface, cm2 Sample volume, mL Pre-treatment of culture plate
(n 4) (n 13) (n 4) (n 5)
20 (n 13) 1 (n 6) 5 (n 11) 10 (n 9) 25 (n 9) B40 (n 8) 40 (n 10) With FCS (n 12) With fibronectin (n 2)
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also pre-treated with fibronectin (Sigma) for 45 min, at a concentration of 2 mg/cm2 of culture surface, in two experiments in order to enhance firm adhesion of the MSC on the culture flask. Non-adherent cells were removed within 24 h of initial plating, with a complete medium change performed once a week thereafter. When layers were confluent (70% confluency), cells were treated with 0.25% trypsin/1 mM EDTA (Invitrogen), counted and replated at a concentration that ranged from 1000 cells/cm2 (n 4) to 50 000 cells/cm2 (n 2) in 25-cm2 flasks for passage 1 (P1) and the passages thereafter.
Immunophenotypic analysis and assessment of functional characteristics of CB MSC The immunophenotypic analysis was performed according to the BM protocol. At day 0 of culture and at subsequent passages, 1 105 cells were stained for 20 min with antiCD45fluoroisothyocyanate (FITC), CD34phycoerythrin (PE), CD29FITC and CD105PE. Labeled cells were thoroughly washed with phosphate-buffered saline (PBS), with 2% FCS and sodium azide, and analyzed on an Epics-Coulter cytometer. For CFU-F assay, at day 0 of culture (the day of initial plating of MNC) 1 105 CB MNC in the standard culture medium described above (low-glucose DMEM with 10% FCS, 200 mM L-glutamine, 10 000 U/mL penicillin and 10 mg/mL streptomycin), without FGF-b, were seeded in each well of a 24-well plate (in triplicate). After 14 days of culture, CFU-F was quantified on an inverted microscope after staining with Giemsa.
Isolation and expansion of BM MSC All the BM samples were cultured under the following conditions. BM MNC, following FicollHypaque (d 1077 g/mL; Lymphoprep-Nycomed) separation, were cultured in a-MEM without nucleotides (Invitrogen), in the presence of 10% FCS (lot selected for optimal growth of MSC; Hyclone Perbio Science), 200 mM L-glutamine and 10,000 U/mL penicillin and10 mg/mL streptomycin (Invitrogen), at a cell concentration of 50 000 cells/cm2 in 25-cm2 T-flasks (Corning), with 1 ng/mL FGF-b (Abcys SA, Paris, France). A complete medium change was performed twice a week. When layers were confluent (85% stroma formation), cells were treated with 0.25% trypsin /1mM EDTA (Invitrogen) and replated at 1000
cells/cm2 (P1) in 25-cm2 T-flasks. MSC were maintained for a number of passages.
Immunophenotypic analysis and assessment of functional characteristics of BM MSC MNC were immunophenotypically characterized by flow cytometry analysis. At day 0 of culture and at various passages (P1P3), 1 105 cells were stained for 20 min with anti-CD45FITC, CD34PE, CD29FITC, CD90 FITC, CD105PE and CD146PE. Labeled cells were washed thoroughly with PBS, with 2% FCS and sodium azide, and analyzed on an Epics-Coulter cytometer. For a CFU-F assay, at day 0 of culture 1 105 BM MNC in the culture medium described above, without FGF-b, were seeded in each well of a 24-well plate (in triplicate). At subsequent passages MSC were plated in 20-cm2 Petri plates at a concentration of 10 cells/cm2 (in duplicate) and, following 14 days of culture, the CFU-F was quantified after staining with Giemsa. The cell doubling time (DT) was calculated using the formula: DT t log(2)/log(cells harvested/cells inoculated); t being the time between initial plating and harvest for the respective passage
Differentiation cultures At P2 or P3, the ability of BM MSC to differentiate into adipocytes (A), osteoblasts (O) and chondrocytes (C) was tested. Adipocyte differentiation MSC at P2 were plated in tissue culture flasks or plates at a cell density of 4 104 cells/cm2 and cultured in DMEM (Gibco BRL, Paisley, UK) supplemented with 20% FCS (Gibco), 100 nM roziglitazone (Cayman Chemicals, Ann Arbor, MI, USA), 1 mM dexamethasone, 60 mM indomethacin and 0.5 mM isobutylmethylxanthine (all from Sigma). The medium was replaced every 34 days for a total of 21 days. Osteocyte differentiation MSC at P2 were plated in tissue culture flasks or plates at a cell density of 1.5 104 cells/cm2 and cultured in DMEM supplemented with 10% FCS, 0.1 mM dexamethasone, 3 mM NaH2PO4 and 0.15 mM ascorbate-2-phosphate (Sigma). The medium was replaced every 34 days for a total of 21 days.
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Chondrocyte differentiation A micropellet culture system was applied in which 2.5 105 cells were centrifuged in a 15-mL polypropylene tube and the pellets cultured in DMEM supplemented with 0.1 mM dexamethasone, 1 mM sodium pyruvate, 0.17 mM ascorbate-2-phosphate, 0.35 mM proline (Sigma), 1 ITS premix (Cambrex Bio Science, Wokingham, UK), 5.35 mg/ mL linoleic acid (Sigma), 1.24 mg/mL bovine serum albumin (BSA) and 10 ng/mL TGF-b1 (R&D Systems, Abingdon, UK). The medium was replaced every 34 days for a total of 14 days. Differentiation was verified by histochemical staining (Oil red O staining for A, Von Kossa staining for O and Alcian blue staining for C) and reverse transciptasepolymerase chain reaction (RT-PCR) analysis (LPL expression for A, ALP and osteoprotegerin expression for O and collagen II expression for C). No differentiation experiments were performed for CB MSC due to the low number of cells harvested.
Results Isolation, expansion, immunophenotypic and functional characteristics of CB MSC Optimal culture conditions resulting in CB MSC isolation were found to be: (1) a higher initial concentration (3 106 cells/cm2) compared with both the BM protocol performed in our laboratory and other CB protocols already published; (2) enrichment of the culture medium with selected FCS (20% of the total volume compared with 10% of the BM protocol); (3) the addition of FGF-b at a higher concentration of 5 ng/mL compared with BM (1 ng/mL in the BM protocol); (4) pre-treatment of the culture surface with FCS; and (5) a high initial volume of the sample ( 40 mL). This set of parameters was characterized as optimal because of the achievement of MSC isolation only under the specific conditions combined together (n 8). Twenty-five per cent of the cultured CB samples formed an adherent layer of MSClike cells only under the optimal culture conditions. The isolated MSC were morphologically similar to the ones derived from BM but appeared later in culture because they needed 34 days from the initial MNC plating until confluency (n 2) and 55 days from P1 to P2 (n 1). The proliferative capacity of successfully isolated MSC, determined by the time needed from one passage to another, was low and they were not effectively passaged beyond the second passage, but retained their morphologic, adherent
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cell characteristics as long as they were in culture. CFU-F colony growth was evident in one case at P1. The total number of colonies obtained was 13. The immunophenotypic analysis of CB-derived MSC cells at P1 showed no expression of CD34 and variable expression of CD45 (017.83%), indicative of their hematopoietic origin. The CD105 expression exhibited a wide range of values (4983%), while another mesenchymal-related surface marker, CD29, was expressed at a range from 57.2% in one experiment to 88.5% in the other. No adequate number of MSC was obtained from CB in order to test the differentiation capacity. In every other case of modified culture conditions, apart from the optimal combination, either no adherent cells were isolated or a heterogeneous bi-morphic adherent cell population appeared (n 2). This population combined both the spindle-shaped and the round-shaped appearance (Figure 1), appeared 1316 days after initial plating of MNC and could be maintained in culture for up to 8 weeks, but without evidence of further expansion ability. In addition, it could not be successfully passaged and did not possess the immunophenotypic profile characteristic of MSC, as shown by the low expression of CD105 and high expression of hematopoietic markers.
Isolation, expansion, immunophenotypic and functional characteristics of BM MSC All BM samples studied successfully resulted in isolation and expansion of MSC. BM MNC formed an adherent stromal cell layer and reached confluency (P1) in approximately 1790.9 days. BM-derived MSC were adherent cells displaying the characteristic fibroblastic spindle shape. MNC at day 0 of culture expressed the hematopoietic markers CD34 and CD45, while there was low expression of mesenchymal-related markers CD105 (1.8590.3%), CD146 (0.9390.3%) and CD90 (2.69 1.1%). Expression of hematopoietic markers gradually decreased at subsequent passages, whereas expression of CD90 (96.591.47%), CD29 (96.692.7%), CD105 (99.49 0.23%) and CD146 (94.0591.45%) significantly increased. The mean number of CFU-F developed in culture was 42.995 at P1, while an average of 33.695 was obtained at P2. The proliferative capacity, as determined by the calculated cell DT, was 390.3 days at P1 and 4.390.6 days at P2. All BM MSC cultures successfully exhibited tri-lineage differentiation, as determined by histochemical analysis and further confirmed with molecular markers
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Figure 1. (a) Adherent cells from CB MNC in two experiments, with a heterogeneous bi-morphic phenotype that combined both the spindle-shaped and the round-shaped appearance. (b) BM-derived MSC at P2 with the characteristic spindle-shaped appearance.
assessed by RT-PCR (LPL expression for A, ALP and osteoprotegerin expression for O and collagen II expression for C). The comparison of phenotypic, functional and immunophenotypic characteristics of MSC derived from CB and BM is depicted in Table 2.
Discussion The ability of blood cells (peripheral blood or CB) to produce a stroma of mesenchymal cells seems to be restricted compared with the potential of BM MNC under similar culture conditions, possibly because of yet un-
defined culture requirements or the need for mobilization of circulating mesenchymal progenitors that have already migrated, together with hematopoietic cells, from the sites of early hematopoietic activity to BM at the time of labor [25]. In spite of reports on the ability of CB to generate MSC, their efficient isolation and expansion in adequate numbers for clinical use should still be considered controversial. MSC circulate in blood at a much lower concentration than that found in BM, specifically 0.052.8 non-hematopoietic progenitors/106 MNC plated in CB or 02.3 clones/1 108 MNC versus 25 MSC/106 MNC
Table 2. Comparison of phenotypic, functional and immunophenotypic characteristics of MSC derived from CB and BM
Morphology
Time to confluence, days CFU-F Immunophenotype, P1
Differentiation potential
CB
BM
Adherent cells with a spindle- shaped appearance similar to the BM MSC Two experiments with a bi-morphic adherent cell population, both spindle-shaped and round-shaped 3490 (n 2) 13 colonies at P1 (n 1)
Spindle-shaped appearance
CD34, 0% (n 2) CD45, 017.83% (n 2) CD105, 4983% (n 2) CD29, 57.288.5% (n 2) No adequate number of MSC obtained
1790.9 (n 23) 42.995 colonies at P1 (n 23) 33.695 colonies at P2 (n 23) 0% (n 23) 0% (n 23) 99.490.23% (n 23) 96.692.7% (n 23) In 23/23 samples Tri-lineage
Cord blood mesenchymal stromal cells
plated in BM [30,31]. This fact could contribute to the difficulty in isolating and expanding MSC from CB under the same culture conditions used for BM MSC. In the present study, CB MNC gave rise to an adherent cell layer with a mesenchymal-like phenotype and immunophenotypic characteristics similar to the relevant ones of BM-derived MSC at a percentage of 25%, when only the experiments with optimal conditions were taken into consideration. We initially tried to grow MSC from CB MNC using the technique already established in our laboratory for the isolation and proliferation of BM MSC. No expansion of adherent cells with MSC phenotypic characteristics was achieved and, therefore, different culture conditions were tried in order to enhance growth of MSC from CB. MNC were set in culture at a higher concentration than the ones used for BM MSC in the different protocols, the culture medium was enriched in FCS and the culture plates were pre-treated with FCS in order to prevent stable adherence of monocytic cells [29]. All the above parameters, in addition to a net sample volume of 40 mL or more and a high concentration of FGF-b in the culture medium, resulted in enhancement of the efficacy of CB MNC to generate a stroma of MSC in vitro. The scarcity of CFU-F in CB further supports the indications that stromal components are not present (or are present at very low frequencies) in CB. The phenotypic and immunophenotypic characteristics of CB-derived MSC were maintained for two subsequent passages, but the number of cells decreased at each passage and the time interval from the initiation of culture until confluency of the stroma was much longer compared with BM. These findings are in accordance with observations made by other scientists [26,30,32]. In some of our experiments, a heterogeneous bi-morphic adherent cell population that combined both the spindle-shaped and round-shaped appearance was observed, but this population could not be successfully passaged and possessed different immunophenotypic characteristics than MSC. One can speculate that other cells, such as macrophages, lymphocytes and other hematopoietic cells, could inhibit the growth of MSC [33]. Interestingly, according to the literature, even in the studies that report successful growth of MSC-like cells from mononuclear CB cells, MSC were not found in all the CB samples analyzed [2426]. The percentage of CB samples generating MSC, according to different reports, ranges from 23% to 63% [19,27,29,31,32], which contrasts
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with the ability of BM MNC to generate stroma of MSC in all the samples studied under the appropriate culture conditions. Variability in the number of MSC in individual CB units, also confirmed by other investigators [19,27,29,31,32], resulted in a wide range of percentage of successful MSC isolation. Erices et al. [27] and Campagnoli et al. [20] showed that mainly CB from preterm labors generated MSC, while another study by Yu et al. [25] showed that MSC mainly reside in fetal blood with a gestational age of 1626 weeks. In addition, Goodwin et al. [30] showed that MSC-like cells, termed non-hematopoietic progenitors, could be isolated from CB but needed more time to grow in culture compared with their BM counterparts. In agreement with this observation, Yang et al. [31] reported that more than half of the CB harvests that generated MSC-like cells were observed during the third week of the culture period. The investigators mentioned above reported a 23% success rate in isolating MSC, and only 2.2% of them with a high expansion capacity. Chang et al. [32] reported a slower appearance of CB MSC, by12 weeks, and a markedly reduced CFU-F frequency, by one-tenth, compared with BM. The differences observed concerning the success rate of isolating MSC from CB, compared with the 100% success rate for BM MSC, pose questions regarding the efficiency of using CB MSC in the clinical setting. Moreover, fewer CFU-F can be generated from CB MSC than from BM MSC, and different CFU-F assays compared with BM have been used by other investigators in order to induce CFU-F growth [30], otherwise impossible because of the low frequency of MSC in CB [33]. However, it has been reported that, although barely isolated, CB MSC showed the highest proliferative capacity compared with BM and adipose tissue but, strikingly, showed no adipogenic differentiation capacity [19]. It should also be mentioned that there is a report of successful isolation of CB MSC from CB MNC by negative immunodepletion of CD3 , CD14, CD19 , CD38, CD66b and glycophorin A cells and limiting dilution in order to obtain single-cell derived, clonally expanded, MSC [34]. Another work supports the isolation of CB MSC by CD133 selection, as the CD133 cell fraction contains more MSC with high proliferative potential [33]. In contrast, BM MSC from children with benign hematologic diseases, in the present study, proliferated in
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culture and were capable of generating an adherent stroma layer in approximately 2 weeks. Immunophenotypic analysis revealed a population of non-hematopoietic cells with a surface antigen profile characteristic of MSC. The immunophenotypic characteristics of MSC could be maintained throughout subsequent passages, as could their proliferative capacity. In addition, it was shown that BMderived MSC had a differentiation potential for adipocytes, chondrocytes and osteocytes, while no adequate number of MSC was obtained from CB in order to test their differentiation capacity, as has also been reported by other investigators [25]. According to the literature, although both BM and adipose tissue MSC demonstrate a multilineage differentiation capacity, there is a debate regarding whether CB MSC can be differentiated towards the adipogenic lineage [19,32]. In addition, Goodwin et al. [30] did not show an ability of chondrogenic differentiation in the CB MSC that they had isolated. Transplantation of MSC is currently under intense investigation and finding alternative sources of these cells, apart from BM, is mandatory because of the limitations that BM could pose. As effective cellular therapy requires a cell type that can be easily isolated and expanded, the findings of the present work suggest difficulties in considering CB an efficient source of MSC for clinical use, even though is easily accessible. Intense work should be concentrated on the search for alternative MSC sources.
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