Human amnion-derived mesenchymal stem cells promote osteogenic and angiogenic differentiation of human adipose-derived stem cells

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.


Introduction
Effective reconstruction of bone defects resulting from trauma and surgical resection is becoming a major clinical challenge in maxillofacial surgery [1][2][3]. With the development of regenerative medicine, tissue engineering utilizing optimal scaffolds seeded with mesenchymal stem cells has shown a promising potential in generating new bone tissue [4,5]. Human adipose-derived stem cells (HASCs) can be obtained from adipose, a highly abundant tissue and easily accessible pool of stem cells [6][7][8] China). Isolation of HAMSCs was performed following the pancreatin/collagenase digestion method [23][24][25]. HASCs and HAMSCs were cultured in 60-mm plates in αMEM supplemented with 100 U/L penicillin, 100 mg/L streptomycin, and 10% FBS in a humidified atmosphere of 5% CO2 at 37˚C. HUVECs were cultured in 60-mm plates in EBM containing 1% FBS and EGM-2 BulletKit in a humidified atmosphere of 5% CO2 at 37˚C. Cells from passage 3 were used and culture medium was changed every 3 days. The study protocols were approved by the Ethics Committee of the School of Stomatology, Nanjing Medical University, China (NO. PJ2013-037-001). Informed consent was obtained from all the participants enrolled in this study.
The coculture system A transwell coculture system was used to investigate the effects of HAMSCs on HASCs as described previously [26]. HASCs were seeded at an initial cell density of 5×10 4 cells/cm 2 in 6-well culture plates. Transwells were placed in other 6-well culture plates and HAMSCs were seeded at increasing HASCs: HAMSCs ratios (5×10 4 cells/transwell, 10×10 4 cells/transwell and 15×10 4 cells/transwell). HASCs were subjected to a 24 h treatment with serum-free medium following the attachment of the cells (approximately 12 h). After washing with PBS, transwells containing HAMSCs were transferred into the corresponding wells of the 6-well culture plate containing HASCs to create the HASCs/HAMSCs transwell coculture system. HASCs in wells with transwells served as the treatment groups, while HASCs without transwells were used as the control groups.

Flow cytometry
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).

ALP activity and mineralized matrix formation
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.

VEGF quantification and HUVECs tube formation assay
The culture supernatant of control and treatment groups was collected from the in vitro coculture system after 14 days and assayed to measure the level of VEGF. A human VEGF ELISA kit was used to quantify VEGF in medium from HASCs and HASCs/HAMSCs groups, according to the manufacturer's instructions. The measured values were expressed as fold changes over that of the control: HASCs treated without HAMSCs. 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 5×10 4 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.

Statistical analysis
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 p < 0.05 was considered to be statistically significant.

HAMSCs promoted HASCs proliferation
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 (Fig 1). Our previous studies have confirmed that HAMSCs were capable of enhancing HBMSCs proliferation [20]. The present results further showed that HAMSCs could also accelerate HASCs proliferation in the transwell coculture system.

HAMSCs promoted ALP activity and extracellular matrix mineralization in HASCs
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 (Fig 2A and 2C). 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 (Fig 2B  and 2D).

HAMSCs promoted osteogenic and angiogenic markers expression in HASCs
Human RUNX2, OCN, and COL1 gene expression were analyzed by real-time PCR in HASCs after 14 days, with and without HAMSCs ( Fig 3A, 3B and 3C). Significant higher levels of these osteogenic markers expression were observed in HASCs/HAMSCs groups with increased HASCs: HAMSCs ratio. VEGF, a valuable cytokine engaged in blood vessel formation and angiogenesis, was also up-regulated in HASCs cocultured with HAMSCs ( Fig 3D). Western blotting showed the protein expression of RUNX2, OCN, COL1, VEGFR1, and Angiogenin were increased in HASCs/HAMSCs groups (Fig 3E). These results suggested HAMSCs enhanced osteogenic and angiogenic differentiation of HASCs.

HAMSCs promoted angiogenesis and ERK1/2 phosphorylation in HASCs
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 (Fig 4A). The HUVECs tube formation assay demonstrated that the addition of culture supernatant from HASCs/HAMSCs groups significantly increased tube structures formed by HUVECs. Extensive vascular networks were observed and increased with HASCs: HAMSCs ratio in HUVECs subjected to culture medium from HASCs/HAMSCs system ( Fig 4C). However, whether the  increased VEGF level is related to the interaction between HASCs and HAMSCs should be studied further.
Our previous studies have indicated that MAPK signal pathway is involved in the HAMSCsdroved osteogenic differentiation of HBMSCs [26]. In addition, it was also reported that MAPK signal pathway played an important role in regulating HASCs osteogenic differentiation [28]. Here, to explore the underlying mechanism, we investigated the effects of HAMSCs on MAPK activation in HASCs. In contrast to HASCs single culture groups, HAMSCs activated the ERK1/2 phosphorylation level in HASCs/HAMSCs groups, while neither the p38 nor JNK phosphorylation appeared to be involved (Fig 4B). These findings demonstrated that HAMSCs enhanced ERK1/2 phosphorylation, which might play a role in modulating HASCs osteogenesis and angiogenesis.

Discussion
The present study identified the valuable effects of HAMSCs on promoting osteogenic and angiogenic potential of HASCs in vitro. We found that HAMSCs promoted proliferation, drove osteogenic differentiation, and enhanced angiogenic via secretion increased VEGF in HASCs. Mechanismly, we demonstrated that ERK1/2-MAPK phosphorylation is associated with the osteogenesis and angiogenesis modulated by HAMSCs, while neither the p38 nor JNK phosphorylation appeared to be involved.
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 [29-31], but most have disadvantages, such as limited availability, high immunogenicity and ethics problems. As an abundant source of stem/progenitor cells, adipose tissue became the focus of considerable interest in regenerative tissue engineering[32]. Among these cells, HASCs gained more attention in recent decades because they can be easily expanded and differentiated into a variety of cells along multiple lineages [33]. Since adipose tissue is abundant and easily access, HASCs exhibit enormous potential for clinical translation into regeneration therapies [34]. Therefore, how to effectively modulate the osteogenic and angiogenic differentiation of HASCs has become an emerging medical problem in bone tissue engineering [35-37].
HAMSCs, isolated from discarded human term placenta, exhibit a potential advantage over other types of MSCs [19]. Our previous findings have showed that HAMSCs were capable of promoting proliferation and osteogenic differentiation of HBMSCs [26], which led us to hypothesize the role of HAMSCs in HASCs-involved new bone formation. The S-phase checkpoints evaluated in HAMSCs, HASCs, and HASCs/HAMSCs groups at 1, 3, and 5 days suggested that HAMSCs promoted HASCs proliferation at early-stage of differentiation. ALP is an early marker of the osteogenic differentiation. Although HAMSCs osteogenesis was much lower than HASCs', treatment of HAMSCs remarkably up-regulated the ALP activity in HASCs. Increased mineralized nodule formation and calcium deposition activity in HASCs cocultured with HAMSCs indicated the positive effects of HAMSCs on late-stage of osteogenic differentiation [38-40]. RUNX2 is a key transcriptional factor associates with several transcription factors and binds to specific nuclear matrix [41]. It is capable of integrating a variety of organize crucial events during the early-stage osteoblastic differentiation of MSCs [42]. OCN is a marker of mature osteogenesis phenotype reflecting calcium deposition during the latestage osteoblastic differentiation [43]. COL1 has been extensively investigated as the most important structural protein of natural bone [44], and its main organic component has been shown to display excellent osteoconductive properties in vitro and in vivo [45,46]. Collectively, the real-time PCR analysis of RUNX2, OCN, and COL1 gene expression suggested HAMSCs motivated HASCs osteogenic differentiation in both early-and late-stage. Additionally, protein expression showed the same variation, which helped us better comprehend HAMSCs' mechanism for action in HASCs-involved bone regeneration.
Bone is a vital organism that needs intraosseous vasculature to maintain normal metabolism [47]. A number of studies have demonstrated that rapid revascularization secrete osteogenic factors is necessary for initiating reliable bone formation in bone regeneration [48,49]. Thus, developing proangiogenic molecules is becoming a key factor in tissue repair.HASCs can secrete a broad range of angiogenic factors, and share equal angiogenic capacity compared with HBMSCs[50]. HAMSCs are capable of acting as stromal cells in the formation, stabilization, and maturation of newly formed vessel [51,52]. Therefore, we hypothesize the effects of HAMSCs in HASCs-involved neovascularization. VEGF, a cytokine engaged in angiogenesis and osteogenesis, evidently enhances new bone formation in animal fracture models [53,54]. In this study, higher level of VEGF secretion was found in HASCs/HAMSCs groups than HASCs groups. HUVECs, the most commonly used type of cells for vessel regeneration studies [55], formed abundant vascular networks by treatment with culture supernatant from HASCs/ HAMSCs groups. In addition, angiogenesis related gene and protein expression also indicated the positive effects of HAMSCs. Thus, these results support HAMSC as activator in HASCsinvolved angiogenesis.
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 [56][57][58], was investigated. Phosphorylation is a remarkable mechanism that modulates the initiating of various factors during osteogenesis and angiogenesis [59][60][61]. Our previous study determined that ERK1/2 phosphorylation was important for the HAMSCs-droved osteogenic differentiation of HBMSCs [62]. Interestingly, the present study also suggested the p-ERK1/2 level was up-regulated by HAMSCs in HASCs. Previous study reported the ERK1/2-dependent RUNX2 activation was an important regulatory mechanism of osteoblast differentiation [63]. In view of the expression of p-ERK1/2 and RUNX2 were both increased by HAMSCs, the ERK1/2-RUNX2 signaling may represent a suitable therapeutic target for promoting HASCs-involved bone regeneration.
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 in vitro at the increased HASCs: HAMSCs ratios. The ERK1/2 phosphorylation level, RUNX2 expression, and VEGF secretion are involved in the underlying mechanism. These findings shed light on the key characteristics of HAMSCs and expand the application of HASCs in bone defects reconstruction.