Comparison of osteogenic differentiation capacity in mesenchymal stem cells derived from human amniotic membrane (AM), umbilical cord (UC), chorionic membrane (CM), and decidua (DC)
Mesenchymal stem cells (MSCs) have been extensively explored as a promising therapeutic agent in the field of bone tissue engineering due to their osteogenic differentiation ability. In this study, the osteogenic differential ability and the effect of fibronectin and laminin on the osteogenic differentiation in four types of MSCs derived from placental tissue are compared to determine the ideal source for bone reconstruction tissue engineering.
The present study examines the osteogenic differentiation levels of four types of MSCs using alizarin red staining and quantifies the calcium levels and alkaline phosphatase (ALP) activity. In addition, this study examines the osteoblast differentiation protein markers osterix, collagen I, osteopontin, and osteocalcin using a Western blot assay. qPCR and EdU labeling assays were employed to identify the kinetics of osteogenic differentiation. Calcium deposit levels, ALP activity, and osteopontin and osteocalcin concentrations were determined to confirm the role of Extracellular matrix (ECM) components role on the osteogenic differentiation of MSCs. The data demonstrated that MSCs isolated from different layers of placenta had different potentials to differentiate into osteogenic cells. Importantly, AM-MSCs and UC-MSCs differentiated into the osteoblast stage more efficiently and quickly than CM-MSCs and DC-MSCs, which was associated with a decrease in their proliferation ability. Among the different types of MSCs, AM-MSCs and UC-MSCs had a higher osteogenic differentiation potential induced by fibronectin due to enhanced phosphorylation during the Akt and ERK pathways.
Taken together, these results indicate that AM-MSCs and UC-MSCs possess a higher osteogenic potential, and fibronectin can robustly enhance the osteogenic potential of the Akt and ERK pathways.
KeywordsMesenchymal stem cells Osteogenic differentiation Fibronectin Kinetics Laminin
mesenchymal stem cells
adipose tissue-derived mscs
bone marrow mscs
Bones have a healing capacity due to their ability to build bone bridges in gaps in response to a minor injury, however, severely damaged bone or critically-sized defects in bone lead to a failure in the gap healing process, and in these cases, bone graft surgical procedures are required . Bone grafts are the second most transplanted tissue in the world behind blood. Despite recent advances in medical care, bone implants do have limitations, such as bad biocompatibility and mechanical integrity . Stem cell-based bone tissue engineering, using a combination of stem cells, biomaterials, and bioactive macro-molecules, is the new frontier for the reconstruction of bone defects . These approaches typically count on the use of stem cells, where stem cells are modified to differentiate to osteoblasts for bone replacement or fill implants.
For years, MSCs have been considered to be multipotent stem cells that are capable of self-renewal and differentiation into a variety of cell types, including osteoblasts, chondroblasts, adipocytes , cardiomyocytes , and pancreatic β cells . MSCs currently can be extracted from different adult and fetal tissues, such as adipose tissue , bone marrow , dental pulp , and placenta tissue . In the early stages of development, adipose tissue-derived MSCs (ADSCs) and bone marrow MSCs (BM-MSCs) have been the paramount agents of clinical cytotherapy. However, two barriers exist to the successful application of these MSCs. First, the isolation of adipose tissue and bone marrow are invasive procedures. Second, ADSCs and BM-MSCs exhibit a significant proliferative rate decline, short life span, and reduced differentiation capacity with increasing age and in several disease phenotypes [11, 12].
Placental tissue can be easily obtained, and usually it is considered as medical waste. It is becoming increasingly appreciated that placenta is the most important source of MSCs. Placental tissue is composed of a variety of tissues, including umbilical cord, amnion, chorion and diaphragm tissues. Recently, several studies have isolated and identified MSCs from placental tissues, including the amniotic membrane (AM) , the umbilical cord (UC) , the chorionic membrane (CM) , and the decidua (DC) . These cells, as stem cells, share the common characteristics of self-renewal, rapid proliferation, and multipotency. Other recent studies, however, have shown different morphologies and multifaceted roles in cytotherapy. Amniotic membrane mesenchymal stem cells (AM-MSCs) have been shown to have a high potential to differentiate into cardiomyocytes and display immunologic tolerance in vivo . Umbilical cord mesenchymal stem cells (UC-MSCs) have been shown to have high proliferative ability and the capacity to differentiate into osteogenic phenotypes . Upon exposure to a neuronal differentiation medium, chorionic membrane mesenchymal stem cells (CM-MSCs) changed their cell morphology and differentiated into neuron like cells . Decidua mesenchymal stem cells (DC-MSCs) have been shown to have an immunomodulatory effect in vivo .
MSCs with osteogenic potential have been extensively studied since 2001 [21, 22]. Introducing ECM components to stem cells-based tissue engineering field not only provide cellular structural support, but also provide the biochemical cues to facilitate and orchestrate cell physiology and phenotype . Placental-derived MSCs (PD-MSCs) are attractive candidates for cell-based bone tissue engineering, but the osteogenic differentiation dynamics and the diversity of PD-MSCs phenotypes during the interaction of extracellular matrix proteins, such as fibronectin (FN) and laminin (LAM), in controlling cell differentiation are worthy of substantial additional investigation.
Given the complexity of MSCs differentiation processes into osteogenic lineages, the current strategies of stem cell-based regeneration for the prevention and/or treatment of human diseases such as bone nonunion, bone defects and osteoporosis, require careful evaluation of the osteogenic differentiation potential between AM-MSCs, UC-MSCs, CM-MSCs, and DC-MSCs.
Osteogenic potential of AM-MSCs, UC-MSCs, CM-MSCs, and DC-MSCs
The osteoblast differentiation protein markers osterix, collagen I, osteopontin, and osteocalcin were examined using a Western blot assay (Fig. 2c). Osterix and collagen I was expressed at higher levels in all of the cell types compared with control cells 21 days after differentiation. The protein level of osteopontin was significantly higher in the AM-MSCs, UC-MSCs, and DC-MSCs than in the control cells, with the exception of the CM-MSCs, which showed no difference between the differentiated and undifferentiated status (Fig. 2d). In addition, the osteogenic marker, osteocalcin, was strongly enhanced in the AM-MSCs and UC-MSCs. Differentiated CM-MSCs and DC-MSCs cells only showed slightly higher levels of protein than the control cells (Fig. 2d). Together, these findings indicate that mesenchymal stem cells divided from different placenta tissue exhibited a unique osteoblast differentiation potential, and there was a significant difference between them regarding their osteogenic differentiation related gene expression.
Kinetics of osteogenic differentiation of AM-MSCs, UC-MSCs, CM-MSCs, and DC-MSCs
Fibronectin and laminin in MSCs differentiation
Fibronectin enhanced osteogenic differentiation via the Akt and ERK pathways
In the early stages of this type of research, efforts had focused on ways to use osteogenic cells, osteoinductive stimulators and biomaterial to develop viable substitutes for bone replacement. Although important advances have been achieved with stem cells and osteoinductive ECM, their applications have not been established largely because of limitations in the choice of cell sources that can efficiently differentiate into osteoblasts and the unpredictability of the complex compounds of the ECM proteins. Therefore, further studies are required to evaluate the differentiation potential of various stem cell sources and the osteoinductive ability of the ECM components.
Recently, several studies regarding osteoblast differentiation from different stem cells have been reported. In particular, Chen et al. showed that AM-MSCs induced in an osteogenic medium had significantly enhanced ALP expression and calcium deposition . Also, it has been shown that UC-MSCs displayed osteogenic differentiation ability on polyglycolic acid (PGA) . CM-MSCs and DC-MSCs also showed osteogenic differentiation potentials . However, no study has evaluated differences in the osteogenic differentiation potentials of AM-MSCs, UC-MSCs, CM-MSCs and DC-MSCs cells. In this study, it was shown that MSCs isolated from different layers of the human placenta had various potentials to differentiate into osteogenic cells. The results of this study showed that AM-MSCs and UC-MSCs had a higher capacity to differentiate into osteoblasts as compared to CM-MSCs and DC-MSCs.
In addition, MSCs differentiation into the osteoblastic phenotype involves several stages, including expression of osteoprogenitor-specific markers, collagen synthesis, and ECM mineralization . Therefore, the current study used not only an alizarin red staining assay, but also a qPCR assay to investigate stage-specific markers produced at different time points in the AM-MSCs, UC-MSCs, CM-MSCs, and DC-MSCs. The qPCR study showed that the AM-MSCs and UC-MSCs displayed a greater osteoprogenitor phenotype than the CM-MSCs and DC-MSCs at day 7, although subcultures of the CM-MSCs and DC-MSCs formed sufficient BMP-6 and Runx2 expression under osteogenic differentiation induction at day 14. Similarly, the AM-MSCs and UC-MSCs exhibited a better osteoblast and osteocyte phenotype than the CM-MSCs and DC-MSCs at days 14 and 21, indicating that the osteogenic potential and extent can be markedly different among cells. Currently, the cellular proliferation rate during the osteogenic differentiation process remains somewhat controversial [29, 30]. In this study, cell proliferation during osteogenic differentiation was assessed using an EdU labeling assay. However, the later stage of osteogenic differentiation induced matrix synthesis and mineralization influent of the EdU and Hoechst 33342 label efficiency. Therefore, the proliferation rate results were only obtained between days 0 and 7. The results revealed that once AM-MSCs and UC-MSCs commit to osteoblasts, they will lose their proliferative ability. However, CM-MSCs and DC-MSCs also lost their proliferative ability even without the committed to osteoprogenitor statue. Pluripotency genes, including OCT4 and SOX2, have shown to be expressed in MSCs and are downregulated upon differentiation, which promoting cell proliferation . However, it remains to be clarified, whether downregulation of pluripotency genes after the osteogenic differentiation induction affect the proliferative ability of CM-MSCs and DC-MSCs.
Laminin  and fibronectin , instead of ECM complex compounds, are the most used compounds to enhance the osteogenic differentiation of stem cell in tissue engineering. In the present study, to evaluate the differentiation level among the AM-MSCs, UC-MSCs, CM-MSCs, and DC-MSCs, osteogenic differentiation markers, including calcium deposits levels, ALP activity, and the osteopontin and osteocalcin concentrations were analyzed. The results indicated that fibronectin, instead of laminin, enhanced the expression of osteopontin and osteocalcin only in AM-MSCs, and UC-MSCs, and it produced a higher ALP activity and calcium deposit levels in all of the cells types. This was consistent with other published findings that fibronectin promotes osteogenic differentiation and cell adhesion than any other ECM protein [34, 35]. Fibronectin seems to be a potential osteoinductive component, however, the molecular signaling pathways that fibronectin mediates to enhance osteogenic differentiation remain to be identified. The current study demonstrates that treatment of AM-MSCs, UC-MSCs, CM-MSCs, and DC-MSCs with fibronectin enhanced the phosphorylation of Akt and ERK. These findings suggest that Akt and ERK activation was associated with fibronectin-induced enhancement of osteogenic differentiation.
Human AM-MSCs, UC-MSCs, CM-MSCs, and DC-MSCs were provided by Sichuan mesenchymal stem cells bank. Cells cultured in α-MEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated FBS (Gibco, Carlsbad, CA, USA), 2 mM l-glutamine (Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco), in a 37 °C, 5% CO2 incubator (Sanyo, Osaka, Japan). Cell growth medium was changed every 3 days. Cells were passaged with 0.125% trypsin (Gibco) at 75% confluence.
To induce osteogenic differentiation of AM-MSCs, UC-MSCs, CM-MSCs, and DC-MSCs, cells were cultured in osteogenic differentiation media with StemPro Differentiation Kit (Gibco) according to the manufacturer’s instructions. AM-MSCs, UC-MSCs, CM-MSCs, and DC-MSCs were seeded in 6-well plates or 96-well plates, osteogenic differentiation media were changed every 3 days. Alizarin Red (Sigma-Aldrich, St. Louis, MO, USA) staining was used to detect calcium deposits.
ALP activity measurement
The ALP activity was measured by quantitative alkaline phosphatase ES characterization kit (Millipore) according to the manufacturer’s protocols. Under alkaline conditions (pH > 10), ALP can catalyze the hydrolysis of p-nitrophenyl phosphate (p-NPP) into phosphate and p-nitrophenol. The amount of p-nitrophenol released is related to the amount of alkaline phosphatase in the reaction. Add p-NPP substrate solution and read the absorbance at 405 nm on a spectrophotometer (Bio-rad, Laboratories, Hercules, CA, USA).
Calcium depos-its quantification
Osteogenesis quantitation kit (Millipore) was used for quantification of calcium depos-its. After alizarin red staining, 10% acetic acid was added to collect the cells and incubated at 85 °C for 10 min. Centrifuge the cell mixture at 16,000g for 20 min. Then, remove the supernatant and adjust pH value to 4.1–4.5 with 10% ammonium hydroxide. Read the absorbance at 405 nm on a spectrophotometer (Bio-rad).
To analyze the accumulative release of osteopontin and osteocalcin, cell culture supernatant was harvested for analyses using ELISA assay kit (abcam). Briefly, 200 μl of cell culture supernatant was added to 96-well plates that were coated with a monoclonal antibody specific to osteopontin or osteocalcin, incubated for 3 h. After washing with PBS, the antibody was added to each well, the plates were incubated for 1 h, washed with wash buffer, and substrate solution was added. Then, the concentration of cytokine was calculated by reading the absorbance at 450 nm on a spectrophotometer (Bio-rad).
RNA extraction and qRT-PCR
During osteogenic differentiation of AM-MSCs, UC-MSCs, CM-MSCs, and DC-MSCs at days 0, 7, 14 and 21, total cellular RNA was extracted by using RNeasy mini kit (Qiagen, Venlo, Netherlands). To remove genomic DNA contamination, DNase I (Invitrogen) digestion was performed. cDNA was synthesized from total cellular RNA using SuperScript III first-strand synthesis system (Invitrogen). Quantitative reverse transcription-polymerase chain reactions (qRT-PCR) reactions were performed using SYBR green master mix (ABI, Invitrogen) and 7300 real-time PCR system (ABI). The mRNA expression levels were normalized using β-actin RNA as internal control. The sequences of primers are shown in Additional file 1: Table S1.
EdU labeling assay
Cells cultured on poly-lysine-coated coverslips in a 24-well plates and incubated at 37 °C for 8 h. 10 μM EdU solution (Invitrogen) was added to the cell culture medium treated for 6 h. Then coverslips were fixed using PBS with 3.7% formaldehyde and permeabilization by a 0.5% Triton X-100 solution. 0.5 ml of Click-it plus reaction cocktail (Invitrogen) was added to each coverslip and incubated for 30 min. Hoechst 33342 (Invitrogen) was applied to show nucleus. Coverslips were preserved with mounting media and imaged by fluorescence microscopy (Leica).
After 21 days osteogenic differentiation of AM-MSCs, UC-MSCs, CM-MSCs, and DC-MSCs, the total cellular protein was extracted using the cell lysis buffer (Beyotime), and concentrations were determined by Bradford protein assay kit (Bio-Rad). Proteins were loaded in SDS-AGE gel and electrophoresed at 80 V for 30 min and 140 V for 60 min. Then, proteins were transferred from gel to nitrocellulose membrane using a trans-blot electrophoretic transfer kit (Bio-Rad). Membranes were blocked in 5% skim milk in TBST buffer for 60 min and incubated with primary antibodies osterix (1:3000, ab94744, abcam 45kd), collagen I (1:3000, ab34710, abcam 125kd), osteopontin (1:2000, ab166709, abcam 65kd), osteocalcin (1:2000, ab93876, abcam 11kd), Tubulin(1:4000, ab4074, abcam 50kd), phosphate-Akt (1:2000, 193H12, Cell signaling technology), Total-Akt (1:2000, C67E7, Cell Signaling Technology), phosphate-Erk1/2 (Thr202/Tyr204, Cell Signaling Technology), total- ERK1/2 (1:2000, Cell Signaling Technology). After washing with TBST buffer, the membranes were incubated with HRP goat anti-mouse IgG (1:3000, Beyotime) or HRP goat anti-rabbit IgG (1:3000, Beyotime). Membranes were then incubated with pierce ECL western blotting substrate (Thermo fisher) and then imaged using chemidoc imaging system (Bio-Rad).
We used the GraphPad Prism software (v7) to conduct statistical analysis (GraphPad Software). Data were expressed as the mean ± SD. Unless otherwise noticed, differences between two experimental groups were applied using an unpaired two-tailed Student’s t-test. For comparison of more than three groups, one-way ANOVA was applied. Results were considered statistically significant with p values: ***p < 0.001**p < 0.01; *p < 0.05.
CYS, SJX, and HZ designed the research. CYS, CY and HZ performed most of the experiments. CY performed the bioinformatic analysis. CY and CYS analyzed the data. CYS, SJX, and HZ wrote the manuscript. All authors read and approved the final manuscript.
We would like to thank Chuan yang and Jiaolian zhu for their kind help and excellent cells culture assistances. We also thank LetPub for its linguistic assistance during the preparation of this manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
The data that support the findings of this study are available from the corresponding author upon request.
Consent for publication
Ethics approval and consent to participate
This work was supported by the National Natural Science Foundation of China (No. 81603478), Natural Science Foundation of Chengdu University of TCM (ZRQN1612), Educational Commission of Sichuan Province (17ZB0149), and Key research and development program of science technology department of Sichuan province (2017SZ0093, 19ZDYF0606).
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