Liang Wang, Zheng-yao Li, Yi-peng Wang*, Zhi-hong Wu, and Bin Yu

Department of Orthopedic Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China

ORIGINAL ARTICLE

Dynamic Expression Profiles of Marker Genes in Osteogenic Differentiation of Human Bone Marrow-derived Mesenchymal Stem Cells△

Liang Wang?, Zheng-yao Li?, Yi-peng Wang*, Zhi-hong Wu, and Bin Yu

Department of Orthopedic Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China

mesenchymal stem cell; gene expression; osteogenesis; osteoblast; differentiation

Objective To observe the expression profiles of osteoblast-related genes in human mesenchymal stem cells (MSCs) derived from bone marrow during osteogenic differentiation.

Methods MSCs were induced to differentiate with MSC osteogenic differentiation medium for 7, 14, 21 and 28 days respectively. Alizarin Red staining was used to detect matrix mineralization. Expression of osteoblast-related genes, including osteocalcin, osteopontin, Runt-related transcription factor 2 (Runx2), alkaline phosphatase and collagen type , was assessed with quantitative reverse transcription I -polymerase chain reaction.

Results On day 14 after induction of differentiation, cells were stained positively with Alizarin Red. The expression levels of these genes exhibited an upward trend as induction time was prolonged. Exposure to osteogenic differentiation medium less than 21 days did not significantly induce osteocalcin expression; osteocalcin expression levels in the differentiated cells induced for 21 and 28 days were 1.63 and 2.46 times as high as the undifferentiated cells respectively (all p＜0.05). Stimulation with MSC osteogenic differentiation medium over 14 days significantly enhanced bone marrow-derived MSCs to express osteopontin and Runx2 genes (all p＜0.05). Osteogenic differentiation medium could significantly induce the expressions of alkaline phosphatase and collagen type genes (all I p＜0.05). Their expressions reached the peak levels on day 21, which were increased more than 4- and 3-fold respectively.

Conclusion Human bone marrow-derived MSCs could exhibit the sequential expression pattern of osteoblast marker genes during osteogenic differentiation in vitro.

Chin Med Sci J 2015; 30(2)：108-113

MESENCHYMAL stem cells (MSCs) are capable of self-renewal and can be differentiated into the osteogenic, chondrogenic, and adipogenic lineages. They are a source of cells for bone and cartilage tissue engineering.1 Among various sources of MSCs, bone marrow has been the commonest one. They have advantages of availability, culture expansion, low immunogenic properties,2 and ease of genetic manipulation, so they have been widely used in numerous clinical applications.3-5 MSCs represent an attractive cell population for bone regeneration due to their expansion properties and osteogenic regeneration potential. Osteogenic differentiation is commonly monitored by sequential expression of known genes. Many studies also presented the significant increase of osteoblast-related genes at a single time point to establish the differentiation, such as day 7 or 14 after differentiation. However, few researches revealed the dynamic change of these marker genes along the differentiation process. An understanding of the dynamic changes of these genes is important for identification and evaluation of different phases of osteogenic differentiation of MSCs.

The purpose of the study is to observe the dynamic change profiles of osteoblast-related genes during the osteogenic differentiation of human bone marrow MSCs. They are regarded as marker genes related to osteogenic differentiation, including osteocalcin (OC), osteopontin (OP), Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and collagen type(Col) genes. TheIIexpression of these marker genes were measured using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for 0, 7, 14, 21 and 28 days during osteogenic differentiation.

MATERIALS AND METHODS

Cell culture

Human bone marrow MSCs (Catalog No. 7500) were purchased from ScienCell Research Laboratories (CA, USA). MSCs were positive for the cell-surface markers CD73, CD90 and CD105. The MSCs were cultured with MSC Medium (MSCM, Catalog No. 7501; ScienCell Research Laboratories) at 37?C in a humidified atmosphere containing 95% air and 5% CO2. The confluent cells were passaged with 0.25% trypsin for up to 3 passages.

Osteogenic differentiation

MSCs (passage 3) were cultured with MSCM in 6-well culture plates. When the cells reached 100% confluence, they were induced to differentiate with MSC osteogenic differentiation medium (MODM, Catalog No. 7531; ScienCell Research Laboratories). The medium was replaced every 3 days. The total RNA was extracted on days 0, 7, 14, 21 and 28 after the initiation of incubation with differentiation-inducing medium. The undifferentiated cells on day 0 were regarded as the control. Three biological replicates were performed for each experiment during osteogenic and chondrogenic differentiation.

Alizarin Red staining

Osteogenic differentiation was confirmed by Alizarin Red staining on day 14. Cells were sequentially washed twice with PBS, fixed with 95% ethanol for 20 minutes, rinsed three times with deionized water and stained by Alizarin Red solution (Genmed Scientific Inc., USA) for 30 minutes at 37?C. After washing four times with deionized water, the stained cells were photographed. Stainings were performed in three replicates.

RNA extraction

Total RNA was extracted using Trizol Reagent (Invitrogen, USA) according to the instructions recommended by the manufacture. The RNA purity and concentration was analyzed with ND-1000 spectrophotometer (NanoDrop Technologies, Inc., USA). The integrity of total RNA was verified to be good by 1% formaldehyde denaturing gel electrophoresis.

qRT-PCR

Total RNA (2 μg) was reversely transcribed into cDNA using a PrimeScript RT reagent kit containing a gDNA Eraser (TaKaRa, Liaoning, China) according to the manufacturer’s instructions.

PCR was performed in a 20 μl of reaction system, including 10 μl SYBR Premix Ex Taq (2x, TaKaRa), 0.4 μl of PCR forward primer (10 μmol/L), 0.4 μl of PCR reverse primer (10 μmol/L), 1 μl of cDNA, and 8.2 μl of double-distilled water, on Thermal Cycler Dice TP800 instrument (TaKaRa Bio Inc., JP). Primers for marker genes were designed and synthesized by Invitrogen (Table 1). RT-PCR reaction was set at an initial denaturation of 10 minutes at 95?C; and followed by 95?C (5 seconds), 52?C (30 seconds), 72?C (30 seconds) in a total 40 cycles and a final extension step at 72?C for 5 minutes. All experiments were performed in triplicate. Transcript values were normalized to GAPDH. Folds change was calculated using 2-??Ctmethods. The relative expression of the transcripts at various time points (including days 7, 14, 21 and 28) was further normalized to day 0 control values to represent a relative fold change of expression.

Table 1. Primers used for polymerase chain reaction

Statistical analysis

Data were analyzed using SPSS 19.0 software. All quantitative data were expressed as mean±standard deviation (SD). Gene expression levels were compared between undifferentiated and differentiated samples. The Student’s t-test was used to compare data between the two groups and one-way analysis of variance (ANOVA) for more than three groups. P<0.05 was considered to indicate a statistically significant difference.

RESULTS

Alizarin Red staining showed matrix mineralization in the cultured cells induced by MODM for 14 days (Fig. 1).

RT-PCR results are shown in Fig. 2. Compared with the undifferentiated cells, exposure to MODM for 7, 14 days increased OC expression in bone marrow-derived MSCs with no statistical significance. Until the 21st day after induction of differentiation, OC expression level began to be significantly higher than that of the control (P<0.05). On day 28, the expression level was 2.46 times that on day 0 (P<0.05). The expression levels of OP and Runx2 genes on day 7 were similar to their counterparts at day 0 with no statistical significance. However, stimulation with MODM for prolonged periods of time significantly enhanced bone marrow-derived MSCs to express OP and Runx2 genes (all P<0.05). On day 28, the expression levels of these two genes increased 7.00-fold and 8.34-fold respectively, compared with the undifferentiated cells (all P<0.05).

Figure 1. Alizarin Red staining of mineralization in human bone marrow-derived mesenchymal stem cells during osteogenic differentiation. (×100)A: control on day 0; B: differentiated cells on day 14.

MODM could significantly induce the expressions of ALP and ColI genes (all P<0.05). Their expression peak appeared on day 21, and slightly decreased on day 28. The ALP expression levels of osteogenic-induced marrowderived MSCs on day 7, 14, 21, and 28 increased 4.29-, 44.87-, 88.41-, and 67.47-fold respectively compared with non-induced MSCs, and ColIexpression levels of the induced cells increased 3.27-, 7.92-, 16.34-, and 9.36-fold on day 7, 14, 21, and 28 compared with non-induced ones (all P<0.05).

DISCUSSION

Stem cells are present in a variety of mesenchymal tissues other than bone marrow and can be isolated from them, such as skeletal muscle,6trabecular bone,7synovium,8adipose tissue,9umbilical cord blood,10and periosteum.11Bone marrow MSCs derive from mesodermal cell lineages with self-renewable capacities and multidirectional differentiation potentials.12,13Their ease of isolation and culture and multi-lineage differentiation potentialmake MSCs suitable and appealing cells in tissue engineering. The cells can differentiate into osteoblasts to support intramembranous bone formation and also into chondrocytes to form cartilage during the initial stages of endochondral ossification.14,15

Figure 2. RT-PCR results of expression levels of OC (A), OP (B), Runx2 (C), ALP (D) and ColI (E) genes.The relative expression levels at different time points were normalized to control values on day 0, to represent the fold change in gene expression.*P<0.05 compared with relative expression levels of marker genes in the undifferentiated cells.

Previous studies on human bone marrow MSCs determined the differentiation of MSCs into osteoblast by observing the formation of calcium nodus and increase of single or two genes expression. However, to date, few studies have continuously observed the expression pattern of osteoblast marker genes after induction of differentiation.

In the process of differentiation into osteoblast, MSCs show the periodic biochemical changes, which involve expression of genes regulating transcription in the early periods and sequential expression of genes associated with the osteogenesis. Hu et al16reported the gene expression during induced differentiation of sheep bone marrow MSCs into osteoblasts. The osteoblasts stagespecifically expressed osteoblast-specific genes such as OP, OC, and ColI.

During the osteogenic differentiation of MSC, three phases of cell proliferation, matrix maturation, and mineralization occur gradually.

OC is synthesized only by mature osteoblast, exhibits the most selective pattern of expression, and is a necessary factor for bone calcification and mineralization.17The expression of OC occurs later during the osteoblast development and contributes to the regulation of the mineral portion of bone. Shui et al18reported that OC mRNA expression was increased in a time-dependent manner, with 1.63-fold increase after 14 days of differentiation. In our study, in the early stage of osteogenic differentiation, the expressions of OC were similar to undifferentiated cells. On day 28, it exhibited 2.46-fold increase than the control group. The results also consisted with previous opinion that OC plays an important role in bone mineralization and calcium homeostasis during the later phase of differentiation. The mRNA of OC regulates the production of the protein, which is the main noncollagenous protein.19

OP, which is produced by osteoblasts during different stages of tissue maturation, involves in bone remodeling.During in vitro bone growth, there is an initial production of OP that continues at a higher level during the mineralization stage.20Although there is no difference between day 7 and day 0, the expression level of OP increased 4-fold at day 14 and reached 7-fold on day 28. Such results also verify by previous study that showed OP expression level continues at a high level during the mineralization stage.20

Runx2 is the earliest known marker of osteogenic differentiation and is required for commitment of mesenchymal progenitors to the osteoblast lineage. It binds to osteoblast-specific cis-acting elements in genes expressed during osteoblast differentiation.21Koch et al22reported that Runx2 gene expression levels were steadily increased between 48 hours and 10 days during osteogenic differentiation. Shui et al18found no major changes of Runx2 level during human osteoblast differentiation. In our study, the expression levels of Runx2 were progressively increased from the beginning. At day 14, the expression was 2.53-fold compared to that of day 0 and reached the peak on day 28 with more than 8-fold.

ALP and Col I were commonly regarded as early markers of osteogenic differentiation and OC was thought to be a later-stage marker.23Col I is essential for mineralization of the bone matrix. It is the most abundant protein in bone matrix and constitutes about 90% of the organic bone matrix and high levels of Col I mRNA expression would be observed during proliferation. In Shui’s study, ALP mRNA expression was increased time-dependently, with more than a 20-fold increase after 14 days differentiation than control culture.18Our results showed that the ALP and Col I exhibited constant high expression levels from the initial. The expression levels of ALP and Col I were more than 4-fold and 3-fold respectively compared with the control at day 7. These expressions peaked at day 21 with more than 80-fold for ALP and 16-fold for Col I. The constant high expression levels along the whole process also verify the two genes as early markers for osteogenic differentiation. The two genes initiate and regulate the synthesis of extracelluar matix from the beginning, which is the core of differentiation.

In addition, Runx2 is regarded as a critical transcriptional factor for osteogenic differentiation and regulates the expression of other osteoblastic genes by binding to the osteoblast-specific cis-acting elements.24It was reported that when active Runx2 levels have been reduced, decreased expression of the genes encoding the main bone matrix proteins including bone OC, OP and Col I were also observed.25In our study, the change trends of expressions of OP, ALP and Col I were consistent with that of Runx2. The relative high expression of Runx2, OP, ALP, and Col I along the whole differentiation process could indicate that these marker genes play important role along the whole process of differentiation.

In summary, our study is an observational research that sequentially observes expression patterns of osteoblast maker genes in human bone marrow-derived MSCs during the process of osteogenic differentiation. By analysis of periodic expression of marker genes of OC, OP, Runx2, ALP and Col I, the alteration of these genes proved to be correlated with the growth and osteogenic differentiation of human bone marrow-derived MSCs. These results may help to understand the detailed expression of related mRNAs and further researches on regulation mechanisms are needed.

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for publication November 24, 2014.

?These authors contributed equally to this work.

Tel: 86-10-69152809, Fax: 86-10-69152809, E-mail: ypwang@medmail.com.cn

△Supported by the National Natural Science Foundation of China (81372007).