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a Department of Orthopaedics and Traumatology, Veterans General Hospital-Taipei, Taipei, Taiwan;
b Department of Microbiology and Immunology, and Immunology Research Center, National Yang-Ming University, Taipei, Taiwan;
c Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan;
d Department of Surgery, School of Medicine, National Yang-Ming University, Taipei, Taiwan
Key Words. Bone marrow stromal cells • Mesenchymal stem cells • Plastic-adherent cells • CD34 negative (CD34-) • Multipotential differentiation
Shih-Chieh Hung, M.D., PhD., Department of Orthopaedics and Traumatology, Veterans General Hospital-Taipei, 201, Sec. 2, Shih-Pai Road, Taipei, Taiwan. Telephone: 886-2-28757557, ext 118; Fax: 886-2-28265164; e-mail: hungsc{at}vghtpe.gov.tw
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ABSTRACT |
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INTRODUCTION |
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MSCs were isolated initially by Friedenstein et al. [1] and subsequently by other investigators [2–8] based on their adherence to tissue culture surfaces. MSCs isolated by this protocol were heterogeneous in morphology and were shown to be multipotential in that they differentiated in culture or after implantation in vivo into osteoblasts, adipocytes, chondrocytes, and possibly muscle cells. After systemic injection, MSCs were incorporated into a variety of tissues, including bone [9, 10], cartilage [9, 10], muscle [11], lung [9, 10, 12], spleen [12], and thymus [12]. Recently, MSCs isolated from human bone marrow have been found to possess the capacity for self-renewal and multilineage potential to differentiate into osteocytic, chondrocytic, and adipocytic lineages [8]. This is the first time that adult human MSCs have been well manipulated ex vivo, and it marks a new era of "stem-cell-based therapy." Stem cells from bone marrow have also been shown to form other kinds of tissues or cells, including hepatocyte [13–15], cardiomyocyte [16–18], neuron, and brain cell [19–23]. It is possible that the entire genome of the adult stem cell has the potential to turn on any aspect of that genome and be any kind of cell. Thus, adult stem cells offer great promise in medicine as they may generate the full spectrum of cell types needed to repair any damaged organ.
Current clinical applications of adult stem cells include the use of allogenic MSCs to treat osteogenesis imperfecta, a disease with a defect in type I collagen [24]. Several animal experiments and clinical trials are now being conducted using MSCs for fracture healing, tendon repair, cartilage regeneration, and support of engrafting after chemotherapy. Generation of brain cells from adult bone marrow demonstrates a remarkable plasticity of adult tissues with potential clinical applications in treating a variety of central nervous system disorders, such as brain injury, stroke, Parkinson's disease, and other neurodegenerative disorders [19–23]. Other potential clinical applications of marrow stem cells include using them to treat hepatic failure [13, 14] and myocardial infarction [16–18]. More recently, marrow stem cells, when transplanted into fetal sheep early in gestation, engrafted and persisted in multiple tissues and underwent site-specific differentiation into chondrocytes, adipocytes, myocytes, cardiomyocytes, bone marrow stromal cells, and thymic stroma [25]. These data support the possibility of the transplantability of marrow stem cells and their potential utility in tissue engineering and cellular and gene therapy applications.
Despite the great interest in MSCs, there is still no well-defined protocol for isolation and expansion of the cells in culture. Most experiments have been conducted using cultures of MSCs that are isolated primarily by their tight adherence to culture plastic dishes, as described by Friedenstein et al. [1]. However, cells isolated in this way were initially heterogeneous and difficult to clone [3, 26]. Several methods have been developed to prepare more homogeneous populations by the use of sorting based on their differences in size [7, 27] or on some specific surface markers [28–31], such as Sca-1 [27] and STRO-1 [32–34], but none of these protocols have gained wide acceptance.
In the present report, MSCs were isolated from human bone marrow aspirates by the use of a unique method that included a specially designed culture device, which was a plastic culture dish comprising a plate with 3-µm pores to sieve out MSCs from bone marrow aspirates. MSCs isolated using this method are a homogeneous population as indicated by morphology and other criteria, such as surface markers. They have the capacity for self-renewal and the multilineage potential to differentiate into osteogenic, adipogenic, and chondrogenic lineages (Fig. 1
). Therefore, we have referred to the cells as size-sieved stem (SS) cells. In addition, if all the cells from each passage had been plated and cultured in our defined conditions, over 1014 SS cells would have been obtained from each 10-ml aspirate in 15 additional weeks of culture.
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METHODS |
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(PDGFR) were purchased from R&D Systems (Minneapolis, MN; http://www.rndsystems.com); epidermal growth factor receptor (EGFR) and CD90 were purchased from Biosource (Nivelles, Belgium; http://www.biosource.com); osteonectin and osteocalcin were purchased from Hematologic Technologies, Inc. (Essex Junction, VT; http://www.haemtech.com); and alkaline phosphatase and type II collagen were purchased from Chemicon Inc. (Temecula, CA; http://www.chemicon.com). The mAb to human STRO-1 was from Dr. Beverly Torok-Storb (Fred Hutchinson Research Center; Seattle, WA; http://www.fhcrc.org). SH2 and SH3 were American type culture collection hybridoma cell lines (ATCC; Rockville, MD).
Bone Marrow Cell Preparation and Cell Culture
Ten-ml human bone marrow aspirates, taken from the iliac crest of normal donors, were diluted 1:1 with phosphate-buffered saline (PBS; GIBCO; Grand Island, NY; http://www.lifetech.com) and centrifuged at 900 g for 10 minutes at room temperature. The washed cells were resuspended in PBS to a final volume of 10 ml and layered over an equal volume of 1.073 g/ml Percoll solution (Sigma; St. Louis, MO; http://www.sigmaaldrich.com). After centrifugation at 900 g for 30 minutes, the mononuclear cells (MNCs) were recovered from the gradient interface and washed with PBS. Percoll fractionated MNCs or nonfractionated bone marrow cells were suspended in Dulbecco's modified Eagle's medium containing 1 g/l of glucose (DMEM-LG; GIBCO) supplemented with 10% fetal bovine serum (FBS; GIBCO), 100 U/ml penicillin, 100 µg/ml streptomycin, and 25 µg/ml amphotericin B. All cells were plated in 20 ml of medium in a culture device (Transwell®; Corning Inc.; Corning, NY; http://www.corning.com) at a density of 106 MNCs/cm2 (Fig. 1
). The culture device was a 10-cm plastic culture dish comprising a plate with 3-µm pores to sieve out MSCs from other bone marrow cells. The cultures were maintained at 37°C in 5% CO2 in air, with an initial medium change at 7 days after initial plating and then medium changes every 3 or 4 days.
Isolation and Expansion of Mesenchymal Cells from Marrow
Two kinds of adherent cells were observed at 3-4 days and 6-7 days, respectively, after seeding Percoll-fractionated MNCs or nonfractionated bone marrow cells into the culture device. The use of Percoll gradient separation of MNCs hindered, rather than improved, the recovery of both kinds of adherent cells. The early adherent cells present in the lower dish were characterized by small size, polygonal shape, and little renewal capacity. The late adherent cells appearing on the upper plate surface had larger size, fibroblastic-like morphology, and the capacity to replicate and are termed here SS cells for convenience. SS cells were harvested with 0.25% trypsin and 1mM EDTA, and replated in a 10-cm plastic culture dish. When SS cells were cocultured again with the small polygonal cells or nonadherent cells, they lost their renewal capacity (data not shown). Once SS cells were more than 80% confluence, they were recovered with trypsin/EDTA and replated at a ratio of 1:3. The expanded cells were used for characterization of their renewal capacity and their specific response to a consistent set of surface marker antibodies.
Flow Cytometry Analysis
The isolated and expanded cells were characterized at passage 2 to 3 by flow cytometric analysis of specific surface antigens. The cells were harvested from the culture dishes by treatment of 5 mM EDTA in PBS. The cells were stained for 30 minutes on ice with fluorescein isothiocyanate- (FITC)- or phycoerythrin- (PE)-conjugated anti-marker mAbs (in optimal concentrations) in 50 µl washing buffer (PBS/1% FBS/0.1% NaN3). Tested markers included hematopoietic lineage markers (CD7, CD14, CD34, CD38, CD45, AC133, HLA-DR), matrix receptors (CD44, CD50, CD54, CD58, CD62E, CD62L, CD62P, CD105), integrins (CD29, CD49b, CD49d, CD51, CD61), factor receptors (tumor necrosis factor receptor 1, TGFßIIR, EGFR, PDGFR), osteogenic precursor markers (STRO-1, alkaline phosphatase, osteonectin, osteocalcin), CD90 (Thy-1), and MSC markers (SH2, SH3). The cell mixture was then washed twice with washing buffer and fixed in 1% paraformaldehyde (in PBS). Cells were analyzed using a fluorescence-activated cell sorter (FACS Vantage SE; Becton Dickinson) using a 525 nm bandpass filter for green FITC fluorescence and a 575 nm bandpass filter for red PE fluorescence.
Induction of Multilineage Differentiation
Cultured cells at passage 2 were nontreated in DMEM-LG supplemented with 10% FBS (as a control) or treated in one of the following formulas: osteogenic differentiation medium: DMEM-LG supplemented with 10% FBS, 50 µg/ml ascorbate-2 phosphate (Nacalai; Kyoto, Japan; http://www.nacalai.co.jp/en), 10-8 M dexamethasone (Sigma), and 10 mM ß-glycerophosphate (Sigma); adipogenic differentiation medium: DMEM-LG supplemented with 10% FBS, 50 µg/ml ascorbate-2 phosphate, 10-7 M dexamethasone, and 50 µg/ml indomethacin (Sigma); or chondrogenic differentiation medium: cell pellets in serum-free DMEM-LG supplemented with ITS+ Premix (GIBCO) and 10 ng/ml TGF-ß1 (Pepro Tech; Rocky Hill, NJ; http://www.peprotech.com). The medium was changed every 3 days, and cells were used for histochemical staining and for immunohistochemistry study after the completion of differentiation by identified morphology.
Histochemical Staining and Immunohistochemistry Study
The medium was removed from the culture, and cells were washed twice with PBS. Cells were fixed in 3.7% paraformaldehyde for 10 minutes at room temperature and washed twice with PBS. The cells treated by osteogenic formula were stained with alkaline phosphatase staining and von Kossa staining to reveal osteogenic differentiation. Those treated by adipogenic and chondrogenic formulas were stained with Oil red-O and Safranin-O or Toludine blue to show adipogenic and chondrogenic differentiation, respectively. Immunohistochemistry for human type II collagen was also done to demonstrate chondrogenic differentiation of the treated cells.
RNA Extraction and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Analysis
Total RNA was extracted from the nontreated (control) and treated cells using the RNeasy Purification Reagent (Qiagen; Valencia, CA; http://www.qiagen.com). Total RNA (1 µg) was reverse transcribed with Mmlv reverse transcriptase for 30 minutes at 42°C in the presence of oligo-dT primer. PCR was performed using specific primers designed from the published sequence of each cDNA as follows: osteopontin (330 bp), sense: 5'-CTAGGCATCAC CTGTGCCATACC-3', antisense: 5'-CAGTGACCAGTTC ATCAGATTCATC-3'; PPAR
2 (352 bp), sense: 5'-GCT GTTATGGGTGAAACT CTG-3', antisense: 5'-ATAAG GTGGAGATGCAGGCTC-3'; col2a1 (399 bp), sense: 5'-CCAGGACCA AAGGGACA GAAAG-3', antisense: 5'-TTCACCAGGT TCACCAGGAT TG-3'; ß-actin (515 bp), sense: 5'-GCACTC TTCCAGCCT TCCTTCC-3', antisense: 5'-TCACCTTCACCGTTCCA GTTTTT-3'. PCR was performed for 30-35 cycles, with each cycle consisting of denaturing at 95°C for 30 seconds, annealing at 55-60°C for 30 seconds, and elongating at 72°C for 1 minute, with an additional 10-minute incubation at 72°C after completion of the last cycle. To exclude possible contamination of genomic DNA, PCR was also applied to reactions without RT. The amplified complementary DNA was electrophoresed through a 1% agarose gel, stained, and photographed under ultraviolet light.
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RESULTS |
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). SS cells maintained uniform morphology and were significantly greater in number than cells that were harvested after seeding marrow cells in dishes only, from which no or few SS cells were harvested. Purification of SS cells was achieved by removal of the nonadherent cells during subsequent changes of medium. SS cells first recovered from the pore-containing plate reached confluence 10 days later (Fig. 2B), and SS cells replated at a ratio of 1:3 reached confluence 7 days later. SS cells maintained normal proliferation and an undifferentiated status during culture expansion even at passage 18.
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) at passage 2 to 3. They were also negative for other markers for hematopoietic cells (CD7 and CD45), and dimly positive for CD14 and CD38. But SS cells were positive for CD90 (Thy-1), a marker for thymocytes and peripheral T lymphocytes. SS cells were negative for markers for some matrix receptors (CD62L and CD62P), dimly positive for CD50, CD54, CD 58, and CD62E, and positive for CD44 and CD105. They were dimly positive for some markers for integrins (CD49b, CD49d, and CD61), but positive for CD29 and CD51. They were negative for some factor receptors (CD120a, TGFßIIR), but dimly positive for EGFR and PDGFR. In contrast with previous reports, SS cells were consistently negative for STRO-1, a marker for osteogenic MSCs. They were also negative for markers for more mature osteogenic precursors (alkaline phosphatase, osteonectin, osteocalcin).
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) or von Kossa stain-positive nodules (Fig. 4), while the controls did not.
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), while the controls did not. Adipogenic induction was also evident with the accumulation of lipid-rich vacuoles within cells with an eccentric deviation of the nucleus.
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), and type II collagen-rich extracellular matrix was evident by immunohistochemistry (Fig. 7).
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shows RT-PCR analysis of the expression of bone, fat, and cartilage-specific genes in nontreated (control) and treated SS cells. Despite the use of equal volumes of RT products, there was some variability in the expression levels of ß-actin, which was used as an internal control. However, specific PCR products of bone (osteopontin), fat (PPAR2), and cartilage (col2a1) were not expressed in the corresponding nontreated cells, but were expressed in osteogenic, adipogenic, and chondrogenic formula-treated cells, respectively. These findings supported the results observed in the histochemical staining and immunohistochemistry study. CBFA-1, a transcription factor of bone, was not a good marker for osteogenesis of human SS cells, as it was expressed in the treated cells and in the controls (data not shown). LPL, a marker for adipogenesis, was also expressed in adipogenic formula-treated cells but not expressed in the controls (data not shown). Col10a-1, a marker for endochondral ossification, was also expressed in chondrogenic formula-treated cells but not expressed in the controls (data not shown).
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DISCUSSION |
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The mechanism involved in isolating human SS cells from a cell mixture relies on the physical characteristics and biological properties of MSCs. The characteristics of MSCs, including their large size [28], their tendency to adhere [1], and their ability to help form hematopoietic colonies in coculture with CD34+ hematopoietic stem cells [39], provide the means to develop a culture device to physically isolate early MSCs. MSCs do not divide until after they are separated from non-MSC cells. Contact inhibition, rather than soluble negative growth factors generated by non-MSC cells, might hinder the proliferation of MSCs. This speculation was supported by the fact that SS cells lost their proliferation capacity only when they were once again in contact with the small adherent polygonal cells and nonadherent hematopoietic cells. In addition to their role in supporting hematopoietic stem cells [29, 39], these results indicated that bone marrow MSCs renew themselves when other bone marrow cells, including the hematopoietic stem cells, have been removed from the culture.
Human SS cells were recovered by seeding Percoll-fractionated MNCs or nonfractionated bone marrow cells into the culture device. It is not necessary to remove red blood cells or to isolate human MNCs from bone marrow to prepare MSCs using this method. The presence of red blood cells in the bone marrow mixture does not disturb the isolation and purification of human bone marrow SS cells because the small red blood cells pass through the pores in the plate to reach the lower culture dish. The separation of MNCs using the Percoll gradient method even resulted in a poor recovery rate of MSCs in our study. Without a need for the procedure of Percoll gradient separation, this is a simple, effective, and economic method to isolate human MSCs.
SS cells isolated in this study had consistent surface molecules as evidenced by more than 30 available marker antibodies. They were negative or dimly positive for CD34 and other markers of the hematopoietic lineage, including CD7, CD14, CD38, and the leukocyte common antigen CD45. Hematopoietic cells were never identified in the population of SS cells. They were also different from marrow fibroblastic cells that remain positive for the CD34 surface antigen [40]. They were consistently negative for STRO-1, a marker for osteogenic MSCs [32–34], and also negative for other markers of more mature osteogenic precursors, including alkaline phosphatase, osteonectin, and osteocalcin [28]. Thus, the results indicated that SS cells were different from the osteogenic MSCs or precursors that are enriched by sorting bone marrow via these mAbs and cannot differentiate into lineages other than bone [29, 41].
Previous reports have made contradictory assertions as to whether cultures of human MSCs are homogeneous. Some have indicated that the cells are heterogeneous in morphology and several other criteria, such as phenotype characteristics and surface molecules [2, 7, 42]. They emphasized the presence of differently shaped and sized cells; the large ones propagated very slowly and the small ones more rapidly. The large cells are referred to as mature MSCs and the smaller ones are referred to as recycling stem (RS) cells [42]. It has been observed that RS cells replicate and give rise to mature MSCs, which become the predominant cell as cultures approach senescence [43]. Almost all the SS cells (>98%) isolated in this study were homogeneous, as indicated by uniform morphology and consistent surface molecules. Whether the variation in cell size or marker expression represented the presence of another lineage of cells or was due to the asynchronized division of SS cells needs to be studied further. SS cells were positive for CD90 (Thy-1), for which RS cells are negative. RS cells were never discerned in SS cells by fluorescence cytometry, size, granularity, or with the more than 30 available antibodies. RS cells may filter through the porous plate and be excluded from the population of SS cells in the current method. Pittenger et al. reported that bone marrow-derived MSCs are homogeneous and positive for CD44, CD62L, CD90, CD120a, and TGFßIIR, but negative for CD14, CD34, CD45, CD49d, and EGFR [8]. SS cells in the present study supported the idea that MSCs are homogeneous, however, they were different from the cells in some surface molecules as reported by Pittenger et al., including CD49d, CD62L, CD120a, TGFßIIR, and EGFR. We speculate that bone marrow includes many groups of MSCs that are different in surface marker analyses.
Recently, it has been shown that human marrow mesodermal progenitor cells (MPCs) cultured on fibronectin with EGF and PDGF BB can be expanded beyond 50 doublings without obvious signs of differentiation or senescence [36]. SS cells cultured in FBS-supplemented DMEM-LG, without the addition of growth factors, can proliferate without differentiation and reach confluence even after 18 passages. SS cells, which express receptors for both the two growth factors used to expand MPCs, could also be manipulated by these two growth factors in future research. The relationship between SS cells and MPCs is currently not clear; it should be interesting to compare these two cells especially after the addition of the two growth factors to SS cells.
In conclusion, human SS cells with relative homogeneity and capacity for renewal and multipotentiality to differentiate were isolated in this study. SS cells may be an ideal source of cells for therapeutic or diagnostic purposes in degenerative or traumatic disorders of mesodermal tissues. Without requiring the use of antibodies to adsorb cells, the method developed here provides an easy, simple, effective, and economic means to isolate and purify MSCs from bone marrow or other MSC sources.
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ACKNOWLEDGMENT |
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REFERENCES |
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