Differentiation analyses of adult suspension mononucleated peripheral blood cells of Mus musculus
- Shahrul HishamZainal Ariffin†1Email author,
- Intan ZarinaZainol Abidin†1,
- Muhammad Dain Yazid1 and
- Rohaya MegatAbdul Wahab2
© Ariffin et al; licensee BioMed Central Ltd. 2010
Received: 7 July 2010
Accepted: 23 October 2010
Published: 23 October 2010
The purpose of this study is to determine whether isolated suspension mouse peripheral mononucleated blood cells have the potential to differentiate into two distinct types of cells, i.e., osteoblasts and osteoclasts.
Differentiation into osteoblast cells was concomitant with the activation of the Opn gene, increment of alkaline phosphatase (ALP) activity and the existence of bone nodules, whereas osteoclast cells activated the Catk gene, increment of tartrate resistant acid phosphatase (TRAP) activity and showed resorption activities via resorption pits. Morphology analyses showed the morphology of osteoblast and osteoclast cells after von Kossa and May-Grunwald-Giemsa staining respectively.
In conclusion, suspension mononucleated cells have the potentiality to differentiate into mature osteoblasts and osteoclasts, and hence can be categorized as multipotent stem cells.
Stem cells, or mother cells, are cells with the ability to divide for indefinite periods in time and to give rise to specific cells. The primitive stage of stem cells can be divided into three types; i.e., totipotent, pluripotent and multipotent cells [1, 2]. Totipotent cells are the most primitive cells, followed by pluripotent cells. The multipotent cell type is the most differentiated type of stem cells . Osteoblasts and osteoclasts are cells that are responsible for bone formation and resorption respectively . During bone formation, osteoblasts deposit the organic and inorganic matrix, whilst osteoclasts remove bone matrix . Osteoblast and osteoclast cells are originated from different lineage, i.e., osteoblasts arise from mesenchymal stem cells and osteoclasts originate from haematopoietic stem cells .
The objective of this study is to determine the potentiality of isolated suspension mononucleated cells to differentiate into osteoblast and osteoclast cells. Osteoblasts were originated from different lineage (mesenchymal stem cells) but osteoclasts were originated from haematopoietic stem cells. This indicates that the isolated mononucleated cells capable to differentiate into more than one type of cells. Our results show that suspension mononucleated cells isolated from peripheral mouse blood have the potential to develop into more than one type of mature cell. The results also demonstrate the plasticity of adult stem cells isolated from peripheral blood. These cells are capable of fully differentiating into osteoblast and osteoclast cells. The presences of osteoblast and osteoclast cells were determined using molecular biology, cells activity and morphology analyses.
Results and Discussion
Proliferation of Mononucleated Cells
The heterogenic populations were characterised using cell culture selection. After 15 days cultured in selection medium, majority of differentiated and precursor cells died due to short lifespans, e.g., granulocytes (30-40 minutes in the peripheral blood with a total lifespan of 7-13 days that varied under certain pathological conditions) , monocytes (5-7 days)  and platelets (3-5 days) . As a result, only primitive mononucleated cells (stem cells) survive after 15 days cultured in selection medium.
Besides differentiation potential, self-renewal is the main property of stem cells. The suspension mononucleated cells population doubling times are 3.1 days . Passages were done every doubling time i.e., 3-4 days. Within 15 days, 4-5 passages were used for each isolates which produce a total cell number of approximately 3-5 × 106 cells/mL for every isolates which start from approximately 1 × 105 cells/mL. As conclusion, within 15 days the suspension mononucleated cells were shown to proliferate in in vitro conditions.
Activation of Molecular Markers in Differentiated Mononucleated Cells
Previous studies have demonstrated that there are several specific gene markers for osteoblast cells, such as osteopontin, alkaline phosphatase, osteocalcin and collagen type I (Col1) [11, 12]. In this study, molecular analysis was performed to determine in vitro differentiation of peripheral blood mononucleated cells into mature osteoblast cells after induction by differentiation factors, i.e., ascorbic acid and β-glycerophosphate, for 14 days.
In this study, the housekeeping gene Gapdh was used as a positive control for mononucleated cells in both types of medium, i.e., proliferation and differentiation medium. Figure 1A shows that RT-PCR amplification produces a Gapdh band (~717 bp) from mononucleated cells in both types of media. The activation of the Gapdh gene proves that cells perform basic metabolic processes required for cell survival. An inherent assumption in the use of housekeeping genes is that expression of the genes remains constant in the cells or in the tissues under investigation .
Differentiated osteoclast cells express specific markers, such as tartrate resistant acid phosphatase and cathepsin K [18, 19]. Therefore, the Catk gene will only be activated in osteoclast cells. Molecular analysis was performed to determine whether the isolated mononucleated cells had indeed differentiated into mature osteoclast cells. The activation of the Catk gene has been observed in mononucleated cells induced in vitro for osteoclast differentiation over a period of 10 days. RT-PCR analysis was done on day 10 since mononucleated cells are believed to differentiate into mature osteoclasts at this time point, thus suitable for molecular analysis [20–23].
Osteoblastic and Osteoclastic Cell Activities
Morphology of Differentiated Osteoblast and Osteoclast Cells
Figures 6C and 6D show mononucleated cells have been differentiated into osteoblasts and osteoclasts respectively. After von Kossa staining in mononucleated cells cultured in osteoblast differentiation medium for 14 days, mineral nodules were found deposited in these cytoplasmic cells (Figure 6C). According to Alhadlaq and Mao (2003), von Kossa staining can reveal mineral nodules in chondrogenic and osteogenic cells differentiated from mesenchymal stem cells . Other studies also used von Kossa staining to observe the effect of stimulants on osteoblastic cells by quantifying the formation of mineralization nodules in osteoblasts cultures . Therefore, isolated mononucleated cells cultured for 14 days in osteoblast differentiation medium can differentiate into mature osteoblast cells.
Mature osteoclast cells are large multinucleated cells with 6-12 nuclei. Another morphological feature of osteoclasts is the presence of ruffled border and sealing zone [31, 32]. May-Grunwald-Giemsa staining showed that these bone-resorbing cells contain many nuclei in its homogenous cytoplasm. May-Grunwald with conjuction of Giemsa is a classic haematology staining procedures that was used to stain peripheral blood and bone marrow specimen. This type of staining can differentiate between nucleus and cytoplasm, which nuclei and cytoplasm as in blue and pink-rose colour, respectively. Osteoclast distinct morphological feature is the presence of multinucleus cells which therefore can be detected using May-Grunwald-Giemsa . After May-Grunwald-Giemsa staining of induced mononucleated cells, the nuclei are purple in colour, while the cytoplasm is lighter than the nucleus (Figure 6D). In this study, mononucleated cells cultured in osteoclast differentiation medium for 10 days showed the morphology of osteoclast cells. The large multinucleated osteoclast cells possessed four nuclei stained purple in colour (Figure 6D). Therefore, mononucleated cells have been differentiated into mature osteoclast cells.
Mononucleated cells were shown to differentiate into osteoblast and osteoclast cells in their respective differentiation media, as shown by molecular biology, cell activity and morphology analyses. Osteoblast and osteoclast cells originated from different lineages, i.e., osteoblasts from mesenchymal stem cells and osteoclasts from haematopoietic stem cells. The capability of these mononucleated cells to generate different differentiated cells types indicates the plasticity of these adult stem cells. In conclusion, the ability of suspension mononucleated cells isolated from adult mouse peripheral blood to differentiate into both cells lineages demonstrates that these types of cells can be categorised as multipotent stem cells.
The animal experimental research has followed the animal guidelines provided by Faculty of Science and Technology, Universiti Kebangsaan Malaysia, which is similar to international animal guidelines.
Isolation and Proliferation of Mononucleated Cells
Mononucleated cells were isolated from mice by density gradient centrifugation using Ficoll-Paque™ Plus. The isolated cells were cultured in proliferation medium containing Alpha Minimal Essential Medium (AMEM) with 10% heat-inactivated Newborn Calf Serum (NBCS) and 2% penicillin/streptomycin. The cultures were incubated at 37°C in a fully humidified atmosphere containing 5% CO2. Each experiment was isolated from one mouse. Therefore for three independent experiments generated from our data were isolated from three different mice.
Differentiation of Mononucleated Cells
Suspension mononucleated cells were seeded at 1.0 × 105 cells/mL in 24-well plates for osteoblast and osteoclast differentiation assays. Cells cultured in complete medium were then supplemented with 50 μg/mL ascorbic acid and 10 mM β-glycerophosphate to induce differentiation into osteoblasts, while 50 ng/mL RANKL and 25 ng/mL M-CSF were added to induce osteoclast differentiation. The cultures were maintained at 37°C in a fully humidified atmosphere containing 5% CO2. For control, the same cells were cultured with complete medium without supplementation of growth factors. Mononucleated cells viability in both differentiation and control mediums were assessed using trypan blue.
Molecular Analyses of Differentiated Mononucleated Cells
Peripheral blood mononucleated cells have been isolated from three different mice and RT-PCR was done for each of them. Total RNA was isolated from approximately 1 × 105 cells that produce approximately 1.6 mg/mL of RNA using TRI-Reagent (Sigma, USA) according to the manufacturer's instructions. Absorbance was measured at 260 and 280 nm to give ratio of A260:A280 was 1.8 to 2.0 in range to determine the samples purity. Total RNA was isolated from cells in differentiation medium and then reverse-transcribed using AMV reverse transcriptase and Tfl DNA polymerase according to the Access RT-PCR System protocol (Promega Corporation, USA). The primers sequences used were: 1) Gapdh, forward:
5'CACTCCAATCGTCCCTACA3' and reverse: 5'AAGGTGGAAGAGTGGGAG3', 2) Opn, forward: 5'CACTCCAATCGTCCCTACA3' and reverse: 5'GCTGCCCTTTCCGTTGTT3', and 3) Catk, forward: 5'GGCAGGGTCCCAGACTCCAT3' and reverse: 5'GTGTTGGTGGTGGGCTAC3'.
First strand cDNA synthesis was done at 45°C for 45 minutes. The pre-denaturation was done at 95°C for 2 minutes. The temperature cycle used to produce the second cDNA strand and RT-PCR amplification for Gapdh (denaturation: 94°C, 30 s; annealing: 62°C, 60 s; elongation: 68°C, 60 s), Opn (denaturation: 68°C, 60 s; annealing: 62°C, 60 s; elongation: 68°C, 60 s) and Catk (denaturation: 95°C, 30 s; annealing: 63°C, 60 s; elongation: 68°C, 60 s) genes. After 40 cycles, elongation of the final strand was done at 68°C for 7 minutes. The RT-PCR products were then analysed via electrophoresis of 1% agarose gels at 85 V using a 100-bp DNA marker (Vivantis). Each RT-PCR product was then subjected for sequencing and analysed.
Osteoblastic and Osteoclastic Cell Analyses
Cell activities for mononucleated cells cultured in osteoblast and osteoclast differentiation medium were determined using an osteologic disc. An osteologic disc is a disc coated with a micro layer of calcium phosphate on its surface. The cultured discs were stained using von Kossa staining on specific days, i.e., days 5, 7, 10 and 14 for osteoblast differentiation medium and days 5, 7 and 10 for osteoclast differentiation medium. The cell activities on the discs were observed using an inverted microscope (Olympus, Model: CKX75). Images were collected using a digital camera.
Morphology Analyses of Differentiated Osteoblast and Osteoclast Cells
Cytospin was used to analyze osteoblast and osteoclast morphology in this study. Approximately, 5-10 × 105 cells/mL mononucleated cells were centrifuged at 78 × g for 5 minutes. The pellet was smeared onto a glass slide and left to air-dry for about 1 to 2 hours. The cells were then stained with von Kossa and May-Grunwald-Giemsa stains to identify osteoblast and osteoclast cells, respectively. The slides were observed using an inverted microscope (Olympus, Model: CKX75). The percentages of differentiated cells were calculated based on a total of 200 cells counted randomly for approximately 5 different fields under microscope for each experiment.
List of abbreviations
Alpha Minimal Essential Medium
Collagen type I
Macrophage Colony-Stimulating Factor
Newborn Calf Serum
Phosphate Buffer Saline
Receptor Activator NF-κB Ligand
Reverse Transcriptase-Polymerase Chain Reactions
Tartrate Resistant Acid Phosphatase.
The authors would like to thank the Universiti Kebangsaan Malaysia for the financial grants (UKM-GUP-BTK-07-15-197 and UKM-OUP-KPB-33-170/2010) during this study.
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