The authors have declared that no competing interests exist.
Tissue engineering using suitable mesenchymal stem cells (MSCs) shows great potential to regenerate bone defects. Our previous studies have indicated that human amnion-derived mesenchymal stem cells (HAMSCs) could promote the osteogenic differentiation of human bone marrow mesenchymal stem cells (HBMSCs). Human adipose-derived stem cells (HASCs), obtained from adipose tissue in abundance, are capable of multi-lineage differentiation. In this study, the effects of HAMSCs on osteogenic and angiogenic differentiation of HASCs were systematically investigated. Proliferation levels were measured by flow cytometry. Osteoblastic differentiation and mineralization were investigated using chromogenic alkaline phosphatase activity (ALP) activity substrate assays, Alizarin red S staining, real-time polymerase chain reaction (real-time PCR) analysis of osteogenic marker expression, and Western blotting. We found that HAMSCs increased the proliferation and osteoblastic differentiation of HASCs. Moreover, enzyme-linked immunosorbent assay (ELISA) and human umbilical vein endothelial cells (HUVECs) tube formation suggested HAMSCs enhanced angiogenic potential of HASCs via secretion of increased vascular endothelial growth factor (VEGF). Thus, we conclude that HAMSC might be a valuable therapeutic approach to promote HASCs-involved bone regeneration.
Effective reconstruction of bone defects resulting from trauma and surgical resection is becoming a major clinical challenge in maxillofacial surgery [
Human amnion-derived mesenchymal stem cells (HAMSCs) can be obtained from human term placenta, a highly abundant tissue and valuable source of stem/progenitor cells[
Trypsin-ethylenediaminetetraacetic acid (EDTA), fetal bovine serum (FBS) and phosphate-buffered saline (PBS) were purchased from Gibco® Life Technologies. The Alizarin red S (pH 4.4), protein assay kit, lysis buffer, ALP and bicinchoninic acid (BCA) assay kits were purchased from the Jiancheng Corp (Nanjing, China). EBM (endothelial basal medium) was purchased from ScienCell® (San Diego, USA). Penicillin G-streptomycin sulfate, α-minimum essential medium (αMEM), dexamethasone, β-glycerophosphate ascorbic acid and dimethyl sulfoxide (DMSO) were purchased from Sigma–Aldrich (St. Louis, MO). Six-well culture plates and transwells (6-Well Millicell Hanging Cell Culture Inserts, 0.4 μm, PET) were purchased from Millipore® (Bedford, MA, USA). Human VEGF ELISA kit was purchased from R&D Systems (Minneapolis, MN, USA). Growth factor-reduced Matrigel was purchased from BD Bioscience (San Diego, CA). EGM-2 BulletKit was purchased from Lonza (Walkersville, MD, USA). The mouse anti-rabbit IgG (L27A9) mAb, anti-mouse IgG (HRP-linked Antibody #7076), phospho-p44/42 (p-ERK1/2) MAPK rabbit mAb, p44/42 MAPK (ERK1/2) rabbit mAb, phospho-p38 (p-p38) MAPK (Thr180/Tyr182) (D3F9) rabbit mAb, p38 MAPK (D13E1) rabbit mAb, RUNX2 (D1L7F) rabbit mAb, phospho-SAPK/JNK (p-JNK) (Thr183/Tyr185) (81E11) Rabbit mAb, SAPK/JNK (JNK) antibody (#9252),and β-Actin (13E5) Rabbit mAb were purchased from Cell Signalling Technology. The anti-Osteocalcin (OCN) antibody (ab133612), anti-Collagen I (COL1) antibody (ab6308), anti-VEGF Receptor 1 (VEGFR1) antibody (ab32152), and anti-Angiogenin (ab10600) were purchased from Abcam. Other reagents used were of the highest commercial grade available.
HASCs were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). HUVECs were purchased from the China Infrastructure of Cell Line Resources (Beijing, China). Isolation of HAMSCs was performed following the pancreatin/collagenase digestion method[
A transwell coculture system was used to investigate the effects of HAMSCs on HASCs as described previously[
The effects of HAMSCs on HASCs proliferation was measured by flow cytometry at 1, 3 and 5 days. Briefly, after starvation in serum-free medium for 24 h, HASCs were washed with PBS. Transwells containing HAMSCs were moved into the corresponding wells of the 6-well culture plate containing HASCs, and the medium was replaced with culture medium containing 10% FBS. HASCs were harvested at day 1, 3 and 5 and fixed with 75% ice-cold ethanol at 4°C for 30 min in the dark. DNA content was measured by a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and the cell cycle fractions (G0, G1, S, and G2 M phases) were processed using CellQuest Pro software (BD Biosciences). Data was analyzed by ModFitLT 3.2 (verity software house, USA).
After transwells containing HAMSCs were moved into the corresponding wells of the 6-well culture plate containing HASCs, both the cell types were cultured in osteogenic medium (OS) containing 10 mM β-glycerophosphate, 100 nM ascorbic acid, and 100 nM dexamethasone. HAMSCs, HASCs, and HASCs/HAMSCs groups were subjected to ALP activity assays and Alizarin red staining. ALP activity assay was performed using an ALP assay kit according to the manufacturer’s protocols at 7 and 14 days. Alizarin red staining was performed at day 21 using 40 mM Alizarin red S (pH 4.4) for 10 min at room temperature. Following rinsing with PBS, mineralized nodules were visualized using an inverted microscope (Carl Zeiss AG, Oberkochen, Germany) and 10 images were captured for each group.
Total RNA was isolated from HASCs in the control and treatment groups by using trizol reagent, according to the manufacturer’s instructions. The RNA was reverse-transcribed into cDNA in a 20-μL reaction by using a PrimeScript RT Master Mix kit. Real-time reverse transcription-PCR was performed with a SYBR Green PCR kit (Toyobo, Osaka, Japan) and ABI 7300 Real-time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). Specific primers were designed as follows: human RUNX2 (forward,
After three washes with cold PBS, total protein was extracted from cells using lysis buffer. The proteins (10 μg) were resolved using 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes, which were blocked with 5% nonfat milk in PBS containing Tween-20 (PBS-T) for 2 h at room temperature. The membranes were incubated at 4°C overnight with primary antibodies specific for RUNX2 (1:1000), OCN (1:1000), COL1 (1:500), VEGFR1 (1:1000), Angiogenin (1:500), ERK1/2(1:500), p-ERK1/2(1:500), p38 (1:1,000), p-p38 (1:1,000), JNK (1:500), and p-JNK (1:500). After three washes with PBST (0.5% Tween 20 in PBS), the membranes were incubated with the relevant secondary antibodies (1:1000) for 1 h at 37°C, washed and visualized by Immobilon Western Chemiluminescent HRP Substrate (Millipore) and visualized using the ImageQuantLAS 4000 mini imaging system (General Electrics, USA). Three independent trials of each experiment were carried out. β-actin (1:500) served as an internal control.
The culture supernatant of control and treatment groups was collected from the
HUVECs were subjected to the culture supernatant from control and treatment groups to assay the formation of tube-like structures. Six-well culture plates were coated with Matrigel according to the manufacturer’s instructions. After HUVECs were incubated in EBM containing 1% FBS and EGM-2 BulletKit for 6 h and plated onto the layer of Matrigel at a density of 5×104 cells/well, medium was replaced by the culture supernatant from HASCs and HASCs/HAMSCs groups. Matrigel cultures were incubated at 37 C for 24 h. Following rinsing with PBS, tube formation was visualized using an inverted microscope (Carl Zeiss AG, Oberkochen, Germany) and representative network of formed tube structures was randomly photographed five shots per each group.
All the quantitative results were performed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Data are presented as the mean ± standard deviation from at least three separate experiments. The Student’s t-test was used to compare data between the two groups. A value of
Cell cycle fractions (G0, G1, S, and G2M phases) were determined by flow cytometry at 1, 3, and 5 days to measure the proliferation of HASCs seeded in the transwell coculture system. There was no significant difference between the proliferation level of HAMSCs and HASCs. However, a statistically increase of S-phase checkpoints with HASCs: HAMSCs ratios in coculture groups compared with the single-culture groups was detected (
The cell cycle fractions (G0, G1, S, and G2 M phases) of HASCs cultured with or without HAMSCs were determined by flow cytometry at 1, 3 and 5 d.
To measure the positive effects of HAMSCs on osteogenic differentiation of HASCs, we investigated ALP activity and extracellular matrix mineralization in HAMSCs, HASCs, and HASCs/HAMSCs groups at different time points. We found that although HAMSCs osteogenesis was much lower than HASCs’, the ALP activity gradually increased in the treatment groups with HASCs: HAMSCs ratio at day 7 and 14, indicating HAMSCs up-regulated the osteoblastic differentiation of HASCs (
The HAMSCs, HASCs, and HASCs/HAMSCs culture surfaces stained positively for extracellular matrix was measured after 21 days. HASCs formed more mineralized matrix compared with HAMSCs. Moreover, increased level of mineralization in HASCs/HAMSCs groups was observed after 21 days in comparison with the HASCs single-culture groups. These observations demonstrated that HAMSCs positively promoted the mineralization in HASCs (
Human RUNX2, OCN, and COL1 gene expression were analyzed by real-time PCR in HASCs after 14 days, with and without HAMSCs (
(A, B, C and D): The mRNA expression of RUNX2, OCN, COL1, and VEGF were analyzed by real-time PCR at 14 d. GAPDH were used as the internal control. (E): Protein expression of RUNX2, OCN, COL1, VEGFR1, and Angiogenin were determined by Western blotting at 14 d, β-actin served as an internal control. *P < 0.05 and **P < 0.01 in contrast to the HASCs groups.
The effects of HAMSCs on angiogenesis in HASCs were further measured by VEGF ELISA assay of the culture medium on day 14 and tube formation assay of HUVECs at 24 h after culture. The HASCs/HAMSCs groups secreted significantly higher level of VEGF than HASCs groups, and the VEGF level gradually increased with HASCs: HAMSCs ratio (
(A): The VEGF level in culture supernatant from HASCs and HASCs/HAMSCs groups was measured by VEGF ELISA assay on day 14. (B): Tube formation from HUVECs was detected at 24 h after culture. (C): Protein expression of p-ERK, ERK, p-p38, p38, p-JNK, and JNK were determined by Western blotting at 14 d, β-actin served as an internal control. Scale bar: 300 μm.*P < 0.05 and **P < 0.01 in contrast to the HASCs groups.
Our previous studies have indicated that MAPK signal pathway is involved in the HAMSCs–droved osteogenic differentiation of HBMSCs[
The present study identified the valuable effects of HAMSCs on promoting osteogenic and angiogenic potential of HASCs
The application of MSCs has been widely designed to promote osteogenesis and angiogenesis for regenerating new bone formation in tissue engineering approaches. HBMSCs, osteoblasts (OB), and dental pulp stem cells (DPSCs) have been used [
HAMSCs, isolated from discarded human term placenta, exhibit a potential advantage over other types of MSCs [
Bone is a vital organism that needs intraosseous vasculature to maintain normal metabolism[
To unveil the underlying molecular mechanisms by which HAMSCs promote HASCs osteogenic and angiogenic differentiation, MAPK signaling, the activation of which is a crucial trigger of MSCs differentiation [
The present study first demonstrates the influence of cocultured HAMSCs in enhancing the osteogenic and angiogenic differentiation of HASCs as well as the potential signal pathway. HAMSCs added to HASCs promoted proliferation, osteogenesis and angiogenesis
(ZIP)
This study was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD; 2014–37)