Human Peripheral Blood Derived Mesenchymal Stem Cells Demonstrate Similar Characteristics and Chondrogenic Differentiation Potential to Bone Marrow Derived Mesenchymal Stem Cells Pan-Pan Chong,1 Lakshmi Selvaratnam,2 Azlina A. Abbas,1 Tunku Kamarul1 1 Tissue Engineering Group (TEG), National Orthopaedic Centre of Excellence for Research and Learning (NOCERAL), Department of Orthopaedic Surgery, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia, 2School of Medicine & Health Sciences, Monash University, Sunway Campus, 46150 Bandar Sunway, Selangor, Malaysia
Received 21 February 2011; accepted 24 August 2011 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.21556
ABSTRACT: The use of mesenchymal stem cells (MSCs) for cartilage repair has generated much interest owing to their multipotentiality. However, their significant presence in peripheral blood (PB) has been a matter of much debate. The objectives of this study are to isolate and characterize MSCs derived from PB and, compare their chondrogenic potential to MSC derived from bone marrow (BM). PB and BM derived MSCs from 20 patients were isolated and characterized. From 2 ml of PB and BM, 5.4 0.6 million and 10.5 0.8 million adherent cells, respectively, were obtained by cell cultures at passage 2. Both PB and BM derived MSCs were able to undergo tri-lineage differentiation and showed negative expression of CD34 and CD45, but positively expressed CD105, CD166, and CD29. Qualitative and quantitative examinations on the chondrogenic potential of PB and BM derived MSCs expressed similar cartilage specific gene (COMP) and proteoglycan levels, respectively. Furthermore, the s-GAG levels expressed by chondrogenic MSCs in cultures were similar to that of native chondrocytes. In conclusion, this study demonstrates that MSCs from PB maintain similar characteristics and have similar chondrogenic differentiation potential to those derived from BM, while producing comparable s-GAG expressions to chondrocytes. ß 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res Keywords: orthopedics; tissue engineering; mesenchymal stem cell; cartilage; cell therapy
The use of cell therapy, that is, autologous chondrocyte implantation (ACI), for the repair of damaged cartilage is well established, demonstrating good short to medium term outcomes. However, it is clear from the many published results reported in many centers conducting clinical trials, there are inherent limitations associated with this technique. These include the low cell yield from the tissues obtained, the invasiveness of the first stage of the procedure required to obtain the needed cartilage and, the site morbidity associated with the procedure. In addition, it has also been described that the use of autologous cells provides limited repair outcomes in the long term owing to their lower potential to undergo cell proliferation.1 An alternative cell sources is therefore needed in order to overcome the many limitations of this technique. It has been previously described that mesenchymal stem cells (MSCs) are able to provide a ready source of chondrocytes, thereby replacing the present technique currently in use. The use of MSCs has been shown to promote superior tissue repair when implanted in focal cartilage defects.2 In contrast to autologous cartilage cells, MSCs have added advantages as they are multipotent (being able to differentiate into various cell lineages) and are expected to have a longer life span, thereby potentially providing enhanced tissue regeneration.3,4 Traditionally, the main source of stem cells has been mainly from bone marrow (BM). In few literatures, BM has been described as the only tissue in the
Correspondence to: Pan-Pan Chong (T: 603-79677548; F: 60379677536; E-mail:
[email protected]) ß 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
body containing a large reservoir of readily available MSCs.5,6 Other alternative sources such as adipose tissue and umbilical cord blood have been previously described albeit with limited availability.7 More recently, MSCs derived from peripheral blood (PB-MSCs) have also been reported, although many researchers have argued that the existence of these cells circulating in adequate numbers remains low.8,9 According to Roufosse et al.,10 when tissue is damaged, MSCs are transported from BM to the damaged site via the circulating PB to promote regeneration. Using this principle, researchers have demonstrated that MSCs can be increased in the peripheral circulation by a technique known as ‘‘blood mobilization.’’ This involves the hyper stimulation of BM production using granulocyte colony-stimulating factor (G-CSF).8 This, however, results in the production of immature progenitor cells, for example, blasts, rather than the true multipotential MSCs.11 In addition, it has also been demonstrated that this technique does not selectively produce MSCs but rather a mix of MSCs, hematopoietic stem cells and, other immature progenitor cells.12 To ensure that therapeutic applications can be performed using MSCs, a large homogeneous population of cells which can differentiate into specific somatic cells, for example, chondrocytes, may be required. This can be achieved by isolating MSCs from a mixed or heterogenous population of stem cells and expanding their numbers using simple cell culture techniques.13 To ensure cells are committed towards a cartilage lineage, chondrogenic induction can be performed in vitro and later be used in transplantation procedures. However, the reproducibility of this method has not been previously described.14 Furthermore, the question also arises as to whether the described method JOURNAL OF ORTHOPAEDIC RESEARCH 2011
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provides the adequate number of cells that are able to retain their ability for cell expression following chondrogenic differentiation. It is hypothesized that MSCs isolated from the blood of normal individuals share the same characteristics and ability to differentiate along chondrogenic lineage as BM-derived MSCs, and that such chondrogenic differentiated MSCs produces extracellular matrix proteins (in particular S-GAG levels) that are comparable to that of chondrocytes. In the present study, we evaluated the feasibility of isolating MSCs from non-mobilized PB-MSCs and confirmed their existence by cellular characterization. The cells were also evaluated for their chondrogenic potential using quantitative and qualitative methods which include immunohistochemistry, spectrophotometry, and gene expression analyses. These results were then compared to BM derived MSCs (BM-MSCs) isolated from the same patients to ensure inter-specimen variations are eliminated.
MATERIALS AND METHODS Harvesting PB and BM Specimens From Human The experiments to use PB and BM derived stem cell were conducted following the approval from the Medical Ethics Committee in University Malaya (reference no.: 369.19). BM and PB specimens were collected after obtaining informed consents from in-patients. BM from 20 individuals (n ¼ 20) were collected using a large aspirator by a dedicated orthopedic surgeons. Samples were obtained from either the femur or tibia of patients undergoing bone grafting or fracture fixation procedures. PB specimens were collected from the patient via venepuncture (through the veins in the upper limb) and collected into vacuum blood collection tubes (EDTA K2). Both PB and BM samples were collected from the same donor at the same time. Culture of Human PB and BM Derived MSCs Three milliliters of Ficoll–Paque PREMIUM (Amersham Biosciences, Uppsala, Sweden) were placed into a 15 ml centrifuge tube. An equal volume of phosphate-buffered saline (Gibco, Carlsbad, CA) was added into the vacuum PB or BM collection tubes. The diluted mixture of specimen was slowly layered on top of the Ficoll–Paque PREMIUM. The centrifuge tube was then centrifuged for 25 min at 2,500 rpm. The mononuclear cells were extracted and transferred into a new 15 ml centrifuge tube. Cells were washed with Dulbecco’s modified Eagle’s low glucose (DMEM) (Gibco) with 1:1 dilution and underwent centrifugation at 1,200 rpm for 10 min. The supernatant was then discarded. Cell pellet formed at the bottom were resuspended using 1 ml of 378C fetal bovine serum (FBS) (Hyclone, Logan, UT). Cells count and viability test were performed. The mixture of mononuclear cells was then cultured in cell culture medium, which consisted of DMEM, 20% FBS, and 1% antibiotic–antimycotic (Gibco). Cultures were maintained at 378C in a humidified atmosphere containing 5% CO2. Suspended cells were discarded after 5–7 days of culture and adherent cells were left to grow on the flask surface. Culture medium was changed every 3 days. PB and BM derived MSCs were characterized using light microscopy to determine cell morphology. JOURNAL OF ORTHOPAEDIC RESEARCH 2011
Immunophenotyping of PB and BM Derived MSCs To characterize and establish the purity of PB-MSCs and BM-MSCs, the following primary monoclonal antibodies (MoAbs) conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCPCY5.5), allophycocyanin (APC) were used: (i) MSC markers: anti-CD29 (integrin b1 chain), anti-CD105 (SH2, endoglin), and anti-CD166 (SB10/ALCAM); (ii) hematopoietic stem cell marker: anti-CD34 (negative control); and (iii) leukocyte marker: anti-CD45 (leukocyte common antigen). To identify surface antigens, cultured cells were washed with PBS, incubated with MoAbs, and later rewashed with PBS again. Thereafter, cells were resuspended in fluorescein-activated cell sorting (FACS) buffer and analyzed using the flowcytometer (FACSCanto, BD Biosciences, Becton, Dickinson and Company, San Jose, CA). All events were collected and quantified using BD FACSDivaTM software (BD Biosciences). Poietics1 human MSCs (Lonza, Walkersville, MD) were used as positive control. Chondrogenic, Osteogenic, and Adipogenic Differentiation To determine the chondrogenic potential of PB- and BMMSCs, cells isolated in previously described technique were subjected to a series of chemically induced chondrogenic differentiation protocol modified from previous published works.15 Cell pellets (approximately 1 million cells) were suspended in alginate solution (1.2% alginate in 150 mM NaCl). One-milliliter suspension was then aspirated into a 5 ml Pasteur pipettes. The cell suspension was dispensed as drops through the Pasteur pipettes into 102 mM CaCl2 solution. The alginate beads were then allowed to polymerize for 10 min, after which they were rinsed twice with 0.9% sterile saline. Chondrogenic induction medium (1 ml) was then introduced into the culture plate together with the beads. The chondrogenic induction medium comprised a serum-free chemically defined medium consisting of DMEM (Gibco), 1% penicillin/streptomycin (Gibco), 2 mM L-glutamine (Gibco), 40 mg/ml ascorbate-2-phosphate (Sigma-Aldrich, St Louis, MO), 1 mM sodium pyruvate (Gibco), 40 mg/ml proline (Sigma-Aldrich, St Louis, MO), 107 M dexamethasone (Sigma-Aldrich), 1 insulin-transferrin-selenium (ITS þ 1) (100) (Gibco), 10 ng/ml transforming growth factor-b3 (TGF-b3) (Gibco), 10 ng/ml fibroblast growth factor-basic (BFGF) (Sigma-Aldrich). The cells were then maintained at 378C in a 95% air and 5% CO2 humidified incubator. Changing of medium was performed every 2–5 days for the duration of the experiment. Chondrogenic MSCs were harvested after 2 weeks of incubation. To investigate the ability of the isolated PB- and BM-MSCs to differentiate into osteoblastic and adipocytic cells, the PB- and BM-MSCs were cultured using a STEMPRO osteogenesis differentiation kit (Gibco) and STEMPRO adipogenic differentiation kit (Gibco) for 14 days. Biochemical Assays to Determine GAG Expressed From Chondrogenic MSCs Chondrogenesis was quantified by measuring sulfated glycosaminoglycans (S-GAG) production using 1,9-dimethylmethylene blue (DMMB) assay. The methods used to process the samples were similar to that described in previous studies16 and, in accordance with the recommended protocol provided by the manufacturer (BlyscanTM Glycosaminoglycan Assay Kit, Newtownabbey, Belfast, Northern Ireland). One million cells were seeded into alginate scaffolds (1 ml) and cultured
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in a 24-well culture plate, which contained 1 ml chondrogenic differentiation medium. On the first, third, and fifth days, 100 ml chondrogenic differentiation medium was collected from cultures for DMMB assay and replaced with 100 ml fresh such media. To ensure that the alginate beads did not provide aberrant readings as a result of dye absorption during chondrogenic differentiation, cell-free alginate beads (in 1 ml) cultured in chondrogenic differentiation medium were used as background control. The Clonetics1 Normal Human Articular Chondrocytes (NHAC-kn) (Lonza) were used as positive controls. Differences among the groups were determined by non-parametric statistical analysis using Mann– Whitney U-test (SPSS software, version 17). Morphological Analysis of Chondrogenic, Osteogenic, and Adipogenic MSCs To determine the histological changes associated with chondrogenesis, PB-MSCs and BM-MSCs were fixed onto smooth surfaced chamber slides (Lab-TekTM Chamber Slides, Thermo Fisher Scientific, Rockford, IL) and stained with Hematoxylin–Fast Green–Safranin O to detect the presence of cartilage matrix proteoglycans expression and cellular localization using light microscopy. In addition to preparations for Hematoxylin–Fast Green staining, cells were counterstained with 0.1% Safranin O for 6 min prior to the dehydration process to produce the Safranin O stained slides. NHAC-kn was compared as positive controls while the undifferentiated MSCs were used as negative controls. After 14 days of cultivation, osteogenic cultures were processed for Alizarin Red S stain, whereas adipogenic cultures were processed for Oil Red O stain. Gene Expression Analysis of Chondrogenic MSCs Total RNA was extracted from the MSCs and chondrogenic MSCs cultured for 14 days using ALLPrepTM DNA/RNA/Protein. RNA was then reverse-transcribed and cDNA was amplified using the appropriate primers. Conditions for reverse transcriptase-polymerase chain reaction and the primers used are described later in this section. Isolation of RNA From the Alginates and cDNA Synthesis The chondrocytes associated pericellular matrix were released by dissolving the alginate in alginate depolymerization solution (sodium citrate, 55 mM; sodium chloride, 0.15 M, pH 6.8 in ultra pure water, Sigma-Aldrich) for 20 min. The cell suspension was then transferred to a centrifuge tube and spun at 1,600 rpm for 10 min at 48C to pellet the freed cells together with the associated pericellular matrices. Total RNA from the chondrogenic MSCs was isolated using ALLPrepTM DNA/RNA/Protein (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The assessment of RNA integrity and concentration were checked using the ExperionTM RNA StdSens Analysis Kit and ExperionTM automated electrophoresis system (Bio-Rad Laboratories, Hercules, CA). Subsequently, 1–2 ml total RNA (100–900 ng/ml) was reverse transcribed into cDNA, using SuperScriptTM III Reverse Transcriptase (Gibco) with Oligo (dT)18 as a primer according to the manufacturer’s instructions. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Analysis Two-microliter aliquots of the resulting cDNA were amplified in a total volume of 50 ml at an annealing temperature of
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588C for 30 cycles, using AdvantageTM 2 PCR Kit (Clontech, CA). The reaction was pre-denatured at 948C for 1 min. Cyclic parameters were denatured at 948C for 15 s, annealed at 588C for 45 s, elongated at 728C for 30 s. Thirty cycles were performed for each primer set. Primer sequence and length of amplified products were: (i) beta-actin (629 bp): sense (50 –30 ): GGC ACC CAG CAC AAT GAA GA and antisense (50 –30 ): GGC ACG AAG GCT CAT CAT TC; (ii) Oct 4 (405 bp): sense (50 –30 ): AGG AGA TAT GCA AAG CAG AA and antisense (50 –30 ): AGA GTG GTG ACG GAG ACA G; (iii) COMP (154 bp): sense (50 –30 ): AGG GAG ATC GTG CAG ACA A and antisense (50 –30 ): AGC TGG AGC TGT CCT GGT AG. Beta-actin was included as a RT-PCR internal control gene. Reaction products were analyzed in ExperionTM DNA 1K Analysis Kit and ExperionTM automated electrophoresis system (Bio-Rad Laboratories).
RESULTS Isolation of MSCs, Cells Expansion, and Cells Growth MSCs were successfully isolated from human BM and PB in all 20 donors. Data for both the BM and PB results obtained from all donors were pooled and presented as the mean and standard deviation for the whole study. From 2 ml of BM or PB harvested, approximately 3–7 million BM-derived mononuclear cells or 1–2 million PB-derived mononuclear cells (Fig. 1E) were isolated respectively. After 10–14 days of culture, cells grew to approximately 80% confluent on the plastic surfaces of T75 cell culture flasks. It was found that approximately 1–2 106 BMMSCs were obtained at the end of passage 0. Isolated BM-MSCs appeared to be more spindle than rounded with many cells demonstrating fibroblastlike morphology (Fig. 1A and B). BM-MSCs were expanded in vitro using the described trypsinization method and sub-cultured up to passage 2. The reseeding cell densities used for the next passage (passage 1) consisted of 2.5 105 cells per T75 cell culture flask. These cells underwent an initial lag phase during the first week before reaching 60–80% confluence. The large adherent cells (fibroblast-like morphology) were found to proliferate faster than the small rounded cells. These observations findings were, however, only of qualitative measure and thus were not substantiated in this study. It was found that for every 2 ml of BM, approximately 10.54 million (0.83 million) MSCs were obtainable at the end of passage 2. In the PB-MSCs cultures, approximately 0.5–1 million surface adherent cells were obtainable from each tissue culture flask after 2 weeks from cell seeding (passage 0) and about 5.37 million (0.60 million) MSCs were obtainable at the end of passage 2. The BM-MSCs demonstrated significantly greater (p < 0.05) cell number as compared to PB-MSCs at passage 2. Unlike the appearance of BM-MSCs, PBMSCs appeared to possess greater number of rounded shaped cells than those in BM-MSCs indicating that JOURNAL OF ORTHOPAEDIC RESEARCH 2011
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Figure 1. Morphology observation of BM-MSCs and PB-MSCs. (A) BM-MSCs at passage 0; (B) BM-MSCs at passage 2; (C) PB-MSCs at passage 0; (D) PB-MSCs at passage 2; (E) mononuclear cells were isolated from PB using Ficoll–Paque PREMIUM method; (F) PBMSCs (passage 2) were isolated after trypsinization.
only limited numbers of MSCs can be isolated from PB as compared to BM (Fig. 1C, D, and F). Immunophenotyping of MSCs Total of 10,000 cells from PB-MSCs, BM-MSCs and Poietics1 human MSCs were each selected for flowcytometry analysis while excluding the debris and non-single cells. Summary of the results is presented in Table 1. JOURNAL OF ORTHOPAEDIC RESEARCH 2011
Biochemical Assays and Morphological Analysis of Chondrogenic-MSCs Proliferation BM-MSCs and PB-MSCs demonstrated the ability to differentiate into cartilage cell type when grown in three-dimensional alginate beads in vitro. S-GAG production measured using the DMMB showed significant glycosaminoglycan expression during the in vitro chondrogenesis of PB-MSCS and BM-MSCs which was comparable to as compared to NHAC-kn. Quantitative
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Table 1. Flow Cytometry Analysis
Combination A Population of gated cells CD29-positive and CD105-positive CD29-positive and CDl66-positive CD105-positive and CD34-negative CD29-positive and CD34-negative CD105-positive and CDl66-positive CDl66-positive and CD34-negative Combination B Population of gated cells CDl66-positive and CD45-negative CD105-positive and CD45-negative CD29-positive and CD45-negative
Control, %
PB-MSCs, %
BM-MSCs, %
71.6 99.9 100 99.9 99.9 100 99.9
94.4 99.8 99.9 99.8 99.4 99.9 99.8
60.8 100 99.9 99.7 99.8 99.9 99.7
72.3 99.9 99.9 99.9
80.2 98.8 97.3 98.2
58.7 99.7 99.8 99.8
Two combinations of surface markers were used: (A) FITC-conjugated CD105, PE-conjugated CD166, PerCP-Cy5.5-conjugated CD34, APC-conjugated CD29; and, (B) FITC-conjugated CD105, PE-conjugated CD166, PerCP-Cy5.5-conjugated CD45, APC-conjugated CD29. MSC makers: anti-CD29 (integrin b1 chain), anti-CD105 (SH2, endoglin), and anti-CD166 (SB10/ALCAM). Hematopoietic stem cells marker: anti-CD34 (negative control). Leukocyte marker: anti-CD45 (leukocyte common antigen). Poietics1 human mesenchymal stem cells (Lonza) were used as positive controls.
assessments demonstrated the highest GAG content in chondrocyte cultures, followed by chondrogenic differentiated BM-MSCs and chondrogenic differentiated PB-MSCs (Fig. 2). There were significant changes over time (Kruskal–Walls test: p ¼ 0.039 for PB-MSCs; p ¼ 0.027 for BM-MSCs; p ¼ 0.027 for NHAC-kn). However, statistical analysis showed no significant difference of s-GAG accumulation in PB-MSCS and BM-MSCs as compared to NHAC-kn (Mann–Whitney U-test: p ¼ 0.055, 0.055, 0.127 for day 1; p ¼ 0.055, 0.055, 0.055 for day 3; p ¼ 0.055, 0.055, 0.513 for day 5). The NHAC-kn showed an abundance of proteoglycan within the extracellular matrix as observed from the Hematoxylin–Fast Green–Safranin O staining histological slides. In the chondrogenic differentiated BM and PB derived MSCs, an increase in Safranin O
Figure 2. Sulfated glycosaminoglycan (S-GAG) production measured from 1,9-dimethylmethylene blue (DMMB) assay. Data obtained from MSCs were treated with chondrogenic medium for Days 1, 3, and 5. Clonetics1 Normal Human Articular Chondrocytes (NHAC-kn) were used as positive controls. Alginate beads cultured in chondrogenic differentiation were used as background controls.
staining indicated good proteoglycan expression within the matrix (Fig. 3). However, the proteoglycan expression in both BM and PB derived MSCs were lower than NHAC-kn. This may due to the proteoglycans being washed away during harvesting of cells from alginate beads. We also found that the binding of Safranin O to proteoglycan was only stoichiometric when the quantity of proteoglycan in the extracellular matrix was high. Therefore, Safranin O may not be a precise marker of proteoglycan content in the extracellular matrix of cartilage. Osteogenic and Adepogenic Induction It can seen that calcium deposits, stained orange-red color were evident in both osteogenic BM-MSCs (Fig. 4E and F) and PB-MSCs (Fig. 4B and C), but not evident in BM-MSCs (Fig. 4D) and PB-MSCs (Fig. 4A). Surprisingly, small lipid containing structures were detected (appeared in red) in PB-MSCs (Fig. 4G and H) and BM-MSCs (Fig. 4J and K) cultured in basal growth medium (DMEM containing 20% FBS). It has been previously documented that serum was a known inducer of adipogenic differentiation.17 However, the small lipid containing structures were not detected in adipogenic PB-MSCs after the 14 days of induction using the commercial adipogenic induction medium (Fig. 4I). Figure 4L demonstrates the lipid vesicle forming adipocytes as seen in adipogenic BM-MSCs. Confirmation of Differential Expression by RT-PCR Total RNA extractions from marrow-derived MSC with a ratio of 28S/18S, equal to or more than 1.7 were used for further experiments. Differential gene expressions of stem cell gene, cartilage-specific gene and internal control were determined using RT-PCR as demonstrated in Figure 5. In the present study, Oct4 (the main binding transcription factor involved in the JOURNAL OF ORTHOPAEDIC RESEARCH 2011
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Figure 3. Microscopic view of Safranin O stains for proteoglycan. In this preparation, proteoglycan is stained orange to red while the cell nuclei is stained black with blue green background. (A) Clonetics1 Normal Human Articular Chondrocytes (NHAC-kn) stained with Safranin O positive. (B) Chondrogenic BM-MSCs stained with Safranin O positive. (C) Chondrogenic PB-MSCs stained with Safranin O positive.
self-renewal of undifferentiated embryonic stem cells) were expressed in undifferentiated BM and PB derived MSCs, but was absent when cells were subjected to chondrogenic differentiation. In contrast, COMP (an important component of cartilage matrix and represents the marker of cartilage turnover), was expressed in chondrogenic MSCs derived from all two sources (BM-MSCS and PB-MSCs) but not in undifferentiated MSCs.
DISCUSSION The present study was conducted to demonstrate that PB derived MSCs have similar characteristics and ability to undergo chondorogenic differentiation as compared to BM derived MSCs, thereby providing an alternative cell source for the repair of damaged cartilage. Despite claims of previous published studies that show the improbability of isolating PB-MSCs from normal circulation,9 our study has demonstrated otherwise. However, as demonstrated in this study, the numbers of MSCs isolated from PB appears to be limited as compared to BM. In the present study, not only was MSCs isolated from PB using our described method, these adherent PB-MSCs have the ability to undergo self-renewal and tri-lineage differentiation equal to that of BM-MSCs. It appears that in the circulation, a small population of CD34 mononuclear cells does exists and is able to proliferate rapidly in cell culture environments as demonstrated in other studies.18 These cells are able to self-replicate and give rise to daughter cells that have the ability to further differentiate into stable committed cells. All of these characteristics are commonly described features of MSCs.19 However, not all MSCs are devoid of CD34. In few studies, it has been demonstrated that in a minority of adult marrow cells, CD34 are frequently being expressed.20 In addition, it has also been described that in 5% of CD34 positive cells, Stro-1 can be identified using monoclonal antibody, which is specific to MSCs.21 Therefore it is sensible to assume that although this study shows that the cells in JOURNAL OF ORTHOPAEDIC RESEARCH 2011
culture to be almost lacking of CD34 positive cells, there is still a possibility that non-MSCs have been cultured together in a MSC predominant cell population. More so when one considers the possibility of other cells, for example, fibrocytes, which exhibit CD34þ and are present in monocyte fractions of human blood, which could potentially mimic MSCs.22 Conversely, one may argue that presence of CD34 observed in our results may have been derived from a small number of MSCs expressing this protein. The use of a combination of CD markers identification was therefore necessary to demonstrate with a high degree of confidence that the cells obtained in this study are more likely to be MSCs. This step was also necessary to ensure that the cells cultured from both PB and BM had fulfilled the criteria for mesenchymal precursors or stem cells, leaving little doubt that the cells isolated were likely to be MSCs.12 The implications of this finding are apparent. Future studies using similar techniques must make provisions to ensure that the data, which may demonstrate the presence or absence of CD34 antigens, are appropriately interpreted. Results in many of these studies demonstrating presences/absence of a combination of CD markers, are however very highly suggestive of MSCs, but not absolute. A more robust and comprehensive analyses using many other identification tools and unique markers, need to be established in order to confirm the presence of MSCs. The presence of MSCs in PB has been demonstrated in previous studies albeit with varying outcomes.8 It is worth noting that in few studies, the culture of PB-MSCs has been only occasionally achieved and not always replicable.23 On the contrary, other studies have demonstrated that MSCs can be isolated from PB repeatedly, however their limitations remains in the low numbers of cells obtained.24 Others have also argued that the cells observed from PB culture are namely fibrocytes, which is one of the two progenitor cells found in PB (also called colony-forming units fibroblastic cells). However, these cells have limited
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Figure 4. Microscopic view of Alizarin Red S and Oil Red O stains. Alizarin Red S: calcium deposits appeared orange-red (A–F); Oil Red O: lipid appeared red (G–L). (A) PB-MSCs in basal growth medium; (B and C) PB-MSCs in osteogenic medium; (D) BM-MSCs in basal growth medium; (E and F) PB-MSCs in osteogenic medium; (G and H) PB-MSCs in basal growth medium; (I) PB-MSCs in adepogenic medium; (J and K) BM-MSCs in basal growth medium; (L) BM-MSCs in adipogenic medium. þ: Positive staining; : negative staining.
growth potential.25 To circumvent this problem, the use of BM mobilization in patients has been described in many literatures.12,26 This technique involves the stimulation of BM using G-CSF treatment followed by the harvesting of progenitor cells from 1 ml of PB subjected to leukapheresis. This method has inherent limitations which include the low yield obtained following BM mobilization, the non-specific mobilization of mixed type of progenitor cells from BM and, the production of
cells which express low MSC-specific gene markers.9,12 The long-term consequence of BM stimulation on donors or patients also remains unknown.26 The report on the success of our ability in isolating MSCs repeatedly from PB would therefore provide greater potential for awide range of clinical implications, eliminating the need for unwarranted risks to patients while providing an important and easily accessible source of cells for tissue engineering.27 JOURNAL OF ORTHOPAEDIC RESEARCH 2011
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futures studies, which should also include a larger number of samples. In summary, this study has shown that the isolation of MSCs may be possible from PB albeit in lower numbers than BM. PB-MSCs have demonstrated to possess similar chondrogenic potential to that of BMMSC. Once chondrogenic differentiation is achieved, PB-MSCs may express similar levels of ECM proteins to that of chondrocytes.
ACKNOWLEDGMENTS This research work was supported by Fundamental Research Grant (FP048/2005D and FP055/2005D) from the Malaysian Ministry of Science, Technology and Innovation. This project is supported by the University of Malaya HIR-MOHE research grant initiative.
REFERENCES
Figure 5. Gene expression in bone marrow derived MSCs (MMSC), blood derived MSCs (BMSC), chondrogenic-MMSC (cMMSC) and chondrogenic-BMSC (cBMSC). L: Ladder (1 kb DNA), 1: Internal control gene (beta-actin, product size: 629 bp), 2: MSC gene (Oct-4, product size: 405 bp), 3: MSC gene (Oct-4, product size: 405 bp), 4: cartilage gene (COMP, product size: 154 bp), 5: cartilage gene (COMP, product size: 154 bp).
Although the use of BM-MSCs showed significantly higher cell number to that of PB-MSCs at the end of passage 2, it was clear that from merely 2 ml of PB, approximately 5 million cells can be expanded in vitro, exceeding that which is required for a number of established repair procedures.28 In addition, PB-MSCs had similar chondrogenic potential to that of BMMSCs making it a good alternative candidate for cell transplantation procedures. While this study does demonstrate that PB-MSCs provide similar chondrogenic expression to that of chondrocyte (through the expressed GAG levels), it is unclear as to whether similar changes may be observed once cells are transplanted in vivo. With increased concerns of possible tumorigenesis manifestation with the use of undifferentiated cells, the use of chondrogenic lineage drive cells may be more readily accepted.29 However, this phenomenon has yet to be proved in any known studies. Several limitations were identified in this study. Restricted resources allowed only one quantitative assessment to be used to determine the chondrogenic protein expression from cells. Other markers should include extracellular protein expression such as aggrecan, collagen type II, and fibronectin. Furthermore, it is only demonstrated the both PB-MSCs and BMMSCs can be differentiated into chondrocytes in vitro while that occurring in in vivo conditions may not be similar. These limitations should be addressed in JOURNAL OF ORTHOPAEDIC RESEARCH 2011
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