Fabrication and Paracrine Effect Research of Bone Marrow-Derived Endothelial Progenitor Cell Sheet

Background: The release of a wide array of endothelial progenitor cell (EPC) sheet-secreted paracrine factors is central to the mechanism by which these cells contribute to tissue repair. The purpose of this study was to fabricate BM-EPC sheet and conduct preliminary investigation on the paracrine effect of SDF-1 (cid:0) about the role of the stromal cell-derived factor-1α (SDF-1α)/CXCR4 axis in the form of tubular structures from BM-EPCs sheet. Methods: EPCs derived from rat bone marrow (BM-EPC) were identied and isolated by the cell-surface markers CD34/CD133/VE-cadherin/KDR using ow cytometry, as well as by dual anity for acLDL and UEA-1. Single-cell suspensions were seeded on temperature-responsive cell culture dishes. After 7 days of incubation, the BM-EPC cells were easily harvested as cell sheets, and a series of biochemical experiments were performed in vitro. The expression levels of SDF-1α/CXCR4 axis-associated genes and proteins were examined using RT ‐ qPCR and western blot analysis, and enzyme-linked immunosorbent assay (ELISA) was applied to determine the concentrations of VEGF, EGF and SDF-1α in the cell culture medium. Results: The BM-EPC cell sheets were successfully harvested. Moreover, BM-EPC cell sheets have superior proliferation and tube formation activity when compare with single cell suspension. When capillary-like tube were formed from EPCs sheets, the releasing of paracrine factors such as VEGF, EGF, and SDF-1 (cid:0) were increasing. To drive the tube formation of BM-EPCs sheets, our mechanism research showed that the activation of PI3K/AKT/eNOS pathway since the phosphorylation of protein CXCR, PI3K, AKT and eNOS were increasing. Conclusion: BM-EPC cell sheets have superior proliferation and tube formation activity that can be used for tissue repair. The strong ability to secrete paracrine factors was be related to the SDF-1α/CXCR4 axis through PI3K/AKT/eNOS pathway. These PI3K/AKT/eNOS


Introduction
The Endothelial Progenitor Cells (EPC) treatment strategy take advantages of preserving or regenerating tissue to develop towards clinical application [1][2][3]. However, the disadvantage of single cells transplantation related aggregation and necrosis make the the feasibility and effectiveness of the approach remain insu cient and di cult in clinical trials [4,5]. EPC derived from bone marrow (BM-EPC) sheet are known to play an important role in angiogenesis by participating not only in the formation of vessels but also in vessel repair and remodeling [6,7]. It can release a wide array of paracrine factors to fabricate tissue repair. However, the forming of BM-EPC sheet and the action characteristics of forming tubular structures remain unclear.
Several studies have demonstrated that SDF-1 expression is su cient to induce EPC cell mobilization and enhance angiogenesis [8]. CXCR4, which is the main SDF-1 receptor, is widely expressed on BM-EPCs.
The SDF-1α/CXCR4 axis has been well documented to play a signi cant role in BM-EPC mobilization and has also been reported to be correlated with the proliferation and survival of EPCs [9,10]. Accumulating evidence has indicated that the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is stimulated to participate in EPC cell proliferation and tube formation capacity. SDF-1α/CXCR4 could also decrease EPC cell apoptosis under serum deprivation or hypoxic conditions via the PI3K/AKT/eNOS pathway [10,11]. Some animal studies indicated that BM-EPC cell sheet technology increased vasculogenesis [12]. However, BM-EPCs cell sheet can not harvested with trypsin and cell-to-cell interactions and the integral structure of the BM-EPCs sheet were unknown [13,14].
Our groups focused on tissue repair for several years and we found tube formation of BM-EPCs sheets was associated with increasing secretion of SDF-1α, EGF and VEGF. VEGF and EGF can acted as paracrine factors to promote BM-EPCs sheets tube forming [15,16], while the effect of SDF-1 was still unknown. Based on the pro-angiogenic effect of SDF-1 [17] and effect characteristics of tube forming from BM-EPCs sheets, we hypothesize BM-EPCs can form tubular structures via the paracrine activity of SDF-1α/CXCR4 axis.

Animals
This study was reviewed and approved by the Animal Ethics Committee of Tianjin First Central Hospital. Adult Wistar rats, 8 weeks of age (weighing 250-280 g), were purchased from the experimental animal center of the Military Academy of Medical Sciences, China. All surgical procedures and care administered to the animals were approved by the Animal Care Committee and performed according to institutional guidelines.

Isolation and culturing of BM-EPCs
Wistar rats were sacri ced by decapitation and soaked in 75% ethanol twice.
Mononuclear cells were isolated from rat BM, especially femur and tibia of the rats, by density gradient centrifugation using Histopaque 1083(Sigma, Stlouis, USA). Isolated cells were resuspended in M199 medium(Gibco, Carlsbad, USA, involved 10% FBS, 20% double antibody, 20 ng/ml VEGF (Sigma), and 1 ng/ml bFGF (Sigma)) and cultured in bronectin-coated dishes and maintained at a 37℃+5% CO 2 incubator. After 4 days in culture, non-adherent cells were removed and fresh medium was added. After the 7 days of culture, BM-EPC cells were identi ed by a combination of speci c surface marker expression.
a. Identi cation of BM-EPCs by endothelial marker Afer 7 days of incubation, the adherent cells were digested by 0.25% trypsin and counted 2×10 6 cells.
Aliquotes containing 1×10 6 cells/100μL into each tubes, were added primary antibody at an appropriate dilution and incubate for 2 h at room temperature. These endothelial markers include CD133 (Proteintech Group, Inc, WuHan, China), CD34 (Proteintech Group, Inc, WuHan, China), VE-cadherin (Bioss, Beijing, China), and KDR (Proteintech Group, Inc, WuHan, China). Then add diluted secondary antibody to the cells and incubate for 1 h at room temperature. The surface markers on BM-EPCs was analyzed by the ow cytometry (BD bioscience, USA), data was analyzed using BD CellQuest ™ Pro software Version 5.1.
Fabrication of BM-EPC cell sheets and thickness assey 1.5×10 6 BM-EPCs were seeded into Nunc UpCell Surface dishes (Thermo Scienti c, USA) coated with vitronectin. Under temperature-responsive culture, incubated for 37℃and 7 days rstly, then reducing the temperature to 25℃ for 30 min, the cells spontaneously detached as contiguous cell sheets and were harvested from the dishes.
To observe detached EPCs sheets thickness, one sheet were xed with 20 mL -20℃ ice-cold methanol solution under 4℃for10 min,3ml primary Anti-Collagen I antibody (1:300,Bioss,Beijing Biosynthesis Biotechnology, Beijing, China) were added to the dishes. After incubation for 5 min at 37℃, 3ml secondary antibody Alexa Flour 488 (1:500, Proteintech Group, Inc, WuHan, China) were injected, incubate at 37℃in the dark for 1 h. Cell nuclei were visualized with DAPI after incubation with secondary antibodies. Images were obtained using uorescence microscope.
BM-EPC cell sheets proliferation assay.
The proliferative capacity of the BM-EPCs sheets was measured using a Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Japan) assay. 5×10 3 /well were seeded into 96-well plates and maintained at a 37℃atmosphere. At the indicated time points (cultivated after 12h,24h,48h and 72h),10 µl Cell Counting Kit-8 was added to each well and cells were cultured for an additional 4 h. Finaly, an enzyme immunoassay analyzer was used to measuring the optical density (OD) at an absorbance wavelength of 450 nm. In order to compare the proliferative capacity of the BM-EPCs sheets, EPCs were used as control.

BM-EPC cell sheets tube formation assay
The BM-EPCs and BM-EPC cell sheets (2 x 10 4 cells) maintained in M199 medium(Sigma-Aldrich, USA) were seeded onto Matrigel(Corning Matrigel Basement Membrane Matrix,Corning,USA)-coated 96-well plates and further cultured in 37℃for 4 h, respectively. To compare the capillary-like tube formation of EPCs sheets and BM-EPCs, Microscope images were captured using inverted phase-contrast microscopy (IX71; Olympus), and the number of network circles for each groups in each image was counted using the Wimasis image analysis program.

Reverse transcription-quantitative PCR (RT-qPCR)
Respectively, total RNA from BM-EPCs and BM-EPCs sheets (1.5×10 6 cells) was extracted using RNAiso Plus (TaKaRa Biotechnology, Japan) according to the manufacturer's protocol. Total RNA was converted into cDNA using PrimeScript TM RT Reagent Kit (TaKaRa Biotechnology, Japan), under the following condition: 37°C for 15 min, 85°C for 5 sec and 4°C for 5 min. cDNA was ampli ed under SYBR Premix Ex Taq TM (TaKaRa Biotechnology, Japan).The sequences of the primers are shown as follows (Table 1) The qPCR thermocycler conditions were as follow rstly 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. β-actin were used as internal reference and relative mRNA expression levels were calculated using the 2 −ΔCt (fold difference), whereΔCt = (Ct of target genes)-(Ct of endogenous control gene, β-action) in experimental samples.
Western blot analysis of CXCR4/p-CXCR4 PI3K/p-PI3K AKT/p-AKT eNOS in BM-EPCs and BM-EPC cell sheets Respectively, total protein was extracted from BM-EPCs and BM-EPCs sheets(1×10 6 cells) and quanti ed using the BCA Protein Assay Kit (TaKaRa, Japan).Proteins lysates were isolated by using 10% SDS-PAGE and then transferred them to PVDF membrane ( Millipore, USA).The membrane were incubated with the antibodies against CXCR4 Detection the expression of paracrine factors SDF-1α, VEGF and EGF using ELISA The BM-EPCs and BM-EPC cell sheets (1x10 6 cells) were incubated in M199 medium which were serumand growth factor-free under 37℃+5% CO 2 atmosphere for 7 d. According to the manufacturer's protocol, the medium was subsequently collected to discover the expression of paracrine factors SDF-1α, VEGF and EGF using ELISA kit. ELISA Kit information is as follows: Rat SDF-1α ELISA kit (Quanzhou Konodi Biotechnology Co. Ltd, China), Rat VEGF ELISA kit (Quanzhou Konodi Biotechnology Co. Ltd, China), Rat EGF ELISA kit (Quanzhou Konodi Biotechnology Co. Ltd, China). The absorbance at 450 nm was measured.
Statistical analysis.
Statistical analyses were conducted using SPSS software version 20.0. Data are expressed as the mean ± standard deviation (SD). Comparisons between two groups were analyzed by a paired samples t-test. Comparisons between groups were analyzed with one-way analysis of variance (ANOVA), following by the least signi cant difference test (LSD-t). p values of less than 0.05 were considered signi cantly different.

Characterization of BM-EPCs
The BM-EPCs were cultured for 7 days and had a cobblestone-like appearance (Figure 1 A, B). After expansion in an incubator for 7 days, BM-EPCs showed the ability to incorporate DiI-acLDL ( Figure 1D) and bind UEA-1( Figure 1C), and these dual-stained cells can be considered to exhibit the proliferative characteristics of BM-EPCs, as presented in Fig 1F. BM-EPCs were identi ed by CD34, CD133, KDR, and VE-cadherin using ow cytometry, as presented in Figure 1G. This information indicated that the cultured cells were BM-EPCs.
Functional analysis of BM-EPC migration toward SDF-1α in a Transwell assay To further con rm the function of BM-EPCs, the directed migration potential of EPCs was detected by a Transwell assay with migration toward SDF-1α. As shown in Figure 2, rat BM-EPCs exhibited directional migration to SDF-1α. Under non-SDF-1α conditions, BM-EPCs were able to migrate through the lter at a low rate of 133.33±4.99 cells per eld of view. As the concentration of SDF-1α in the lower chambers increased, the chemotaxis of EPCs became more obvious (Figure 2 E). There were signi cant differences in the level of SDF-1α between the 10 ng/ml group (180.67±7.72 cells per eld of view) and the other groups ( p=0.0 vs 0 ng/ml group; p=0.009 vs 1 ng/ml group; p <0.05). Compared with 10 ng/mL SDF-1α codition, BM-EPCs, under the 100 ng/ml SDF-1α condition, showed the highest rate of migration through the lter (185.33±9.29 cells per eld of view), but this difference did not reach statistical signi cance(p=0.499).

Gross examination of BM-EPC sheets
After inoculation on temperature-responsive culture plates, the cells spontaneously detached as contiguous cell sheets and were harvested. The gross examination of BM-EPC cell sheets is shown in Figure 3. As shown in the pictures, the EPCs were remarkably connected ( Figure 3A). The EPC sheets were irregular in shape and polygonal ( Figure 3B, C). Under a uorescence microscope, the thickness of the single-layer cell sheet was approximately 15 μm ( Figure 3D). The multilayer cell sheet with approximately 3-4 layers was 60 μm ( Figure 3E).

The cell sheet system accelerates cell proliferation and cell tube formation in vitro
A Cell Counting Kit-8 (CCK-8) assay was used to quantify the levels of cell proliferation. The OD values of the EPC sheet groups were increased compared with those of the EPC groups. In addition, with increasing time within 24 h, the cells in the EPC cell sheet group grew at a signi cantly faster rate than did the cells in the EPCs groups ( Figure 4C). After 24 h, the cells in the EPC sheet proliferated slowly and initiated apoptosis, but the proliferation rate was not obvious in the EPC groups. Using the in vitro model of angiogenesis, we assessed the capacity of the two groups to form tubular structures. After seeding onto Matrigel for incubation for 4 h, the EPC groups exhibited almost no tube formation ( Figure 4B), whereas the EPC sheet groups formed signi cantly more complete tubes ( Figure 4A). Quantitative analysis showed that the total number of tubes formed by the EPC sheet groups (29.33±5.13) was signi cantly greater than that formed by the EPC group (6.67±4.61 and p<0.05).

The expression of CXCR4, PI3K, AKT, and eNOS in EPC sheets
We examined the mRNA expression of genes in the SDF-1α/CXCR4 axis in BM-EPC cell sheets and EPCs by RT-qPCR. RT-qPCR analysis showed that there was no signi cant difference in CXCR4, PI3K, or AKT mRNA levels between the BM-EPC cell sheets and EPCs groups ( Figure 5A, B, C). Interestingly, the level of eNOS expression was signi cantly higher in the BM-EPC cell sheets groups than in the EPCs groups ( Figure 5 D, p=0.001). To explore whether the SDF-1α/CXCR4 signaling pathway plays a positive role in paracrine BM-EPC cell sheets, western blotting was used to analyze the difference in protein expression in the two groups ( Figure 5E-H). The results showed that the phosphorylation of protein CXCR, PI3K, AKT, eNOS was increasing in the BM-EPC cell sheets groups that form tubular structures when compared with the EPCs groups. These protein was located in PI3K/AKT/eNOS pathway, and downstream of the SDF-1α/CXCR4 axis. These results indicated BM-EPC cell sheets form tube was associated with the activation of SDF-1 /CXCR4 axis and PI3K/AKT/eNOS pathway.

BM-EPC cell sheets promoted the paracrine factors VEGF, EGF, and SDF-1α
Furthermore, to detect the differences in paracrine factors between the two groups, we applied ELISA to determine the concentrations of VEGF, EGF and SDF-1α in the cell culture medium (Figure 6 A-C). The results showed that the expression levels of VEGF (194.72±8.00 pg/ml vs 163.07±2.68 pg/ml), EGF (298.33±1.17 pg/ml vs 288.17±1.51 pg/ml) and SDF-1α (5.21±0.02 pg/ml vs 4.84±0.03 pg/ml) protein in the BM-EPC cell sheets groups were signi cantly higher than those in the EPCs groups (p=0.002, p=0.001 and p=0.00, respectively). Based on the paracrine of VEGF and EGF, as well as the activation of SDF-1 /CXCR4 axis, we speculated the form of tubular structures from BM-EPCs sheet was associated with the increasing paracrine effect of VEGF, EGF and SDF-1 .

Discussion
Although the exact de nition of BM-EPC cells remains unclear in terms of speci c surface markers, the vasculogenic, angiogenic, and bene cial paracrine effects of transplanted BM-EPC cells in the treatment of ischemic diseases cannot be overlooked [18].The most widely accepted typical de nition is the coexpression of the cell-surface markers CD34/CD133/VE-cadherin/KDR [19].In the present study, stem cell markers and endothelial cell markers, CD34 + /CD133 + /VE-cadherin + /KDR + , were used to identify and isolate the BM-EPC cells by ow cytometry, as well as dual a nity for acLDL and UEA-1 [20]. The results revealed that the isolated and cultured cells exhibited typical characteristics of BM-EPCs. Moreover, BM-EPCs sheets formed tubular structures in the in vitro model of angiogenesis.
SDF-1α, which mediates many disparate processes exclusively via a single cell surface receptor known as chemokine receptor CXCR4, has been found to promote BM-EPC cell mobilization into the ischemic site, where they promote repair by inducing vasculogenesis and secreting bene cial paracrine factors [8,21,22]. Following veri cation of the migration potential of EPCs, the data from the present study indicated that a dose of 10 ng/ml SDF-1α is the optimal concentration for BM-EPC cell proliferation. The SDF-1α/CXCR4 axis is important for the homing or recruitment of circulating EPCs in response to hypoxia or injury [23]. Recent studies have supported the central role of the SDF-1α/CXCR4 axis in regulating the mobilization of BM-EPC cells and its potential signi cance in cardiac repair through induction of angiogenesis and cardioprotective functions after myocardial infarction [24], as well as several intracellular signaling pathways [25]. Among these signals, the PI3K/AKT pathway plays a very important role by stimulating growth factor production and adhesion molecule interactions, including the activation of endothelial nitric oxide synthesis (eNOS) activity, which has been con rmed to decrease EPC cell apoptosis [11,26].
Cell sheets, whose composition is similar to that of natural tissues, are harvested without enzyme treatment, and cell-to-cell interactions and the sheet structure are well preserved. Compared with traditional cell suspension injection methods, in which most of the cells are washed-out into blood vessels and can may die due to the locally harsh ischemic microenvironment, cell sheet technology has been widely used in regenerative medicine as a novel method of cell therapy [14,27,28]. BM-EPCs cells, which have the advantage of a higher proliferation rate and can facilitate vessel formation and produce pro-angiogenic factors to enhance vascularization after implantation, have been regarded as an ideal candidates to address vascular issues alonely or in cocultured with other cells. Recently, Kawamura et al. reported that MSCs and BM-EPC cells, as colayered cell sheets, prevented cardiac dysfunction and microvascular disease [12]. Another study showed that EPC-SMC bilevel cell sheet technology facilitated the natural interaction between EPCs and SMCs, thereby creating a structurally mature, functional microvasculature in a rodent ischemic cardiomyopathy model, leading to improved myocardial function [29].
Previous research has shown that the release of a wide array of EPC-secreted paracrine factors is central to the role of EPCs in myocardial repair [30,31], however, signaling pathways related to paracrine factors are rarely reported [32]. This limited but valuable information hints at the potential relation between paracrine factor secretion and the SDF-1α/CXCR4 axis. We hypothesized that the SDF-1α/CXCR4 axis might have been activated by a paracrine mechanism, through the PI3K/AKT pathway. In this study, we seeded BM-EPCs in suspension on temperature-responsive cell culture dishes and cultured them for 7 days. After 7 days of incubation, the cultured BM-EPC cells had a polygonal cobblestone shape and were easily harvested as a cell sheet, and a series of biochemical experiments were performed in vitro. We found that the levels of cell proliferation and tube formation in the BM-EPC cell sheet group were remarkably increased compared with those in the single-cell groups. To further explore the signaling pathway involved in the mechanism of paracrine action in vitro, the expression levels of SDF-1α/CXCR4 axis-associated genes and proteins were examined using RT-qPCR and western blot analysis, respectively. We found that the levels of genes associated with the PI3K AKT pathway were not different between the two groups; however, in accordance with previous studies on protein levels of factors associated with this pathway, our present study indicated that the levels of PI3K/AKT/eNOS were all augmented in the BM-EPC cell sheet groups. The data from the present study implied that the SDF-1α/CXCR4 axis may accelerate cell proliferation and decrease apoptosis in the cell sheet system [33,34].
Previous studies suggested that the functional bene ts observed after EPC cell transplantation in experimental animal models of myocardial infarction might be related to the secretion of paracrine factors. These factors include factors that promote neovascularization, anti-in ammation effects, promoting cell migration and proliferation, as well as other known or unknown factors [35,36], such as SDF-1, insulin-like growth factor-1, hepatocyte growth factor (HGF), epidermal growth factor (EGF) VEGF, and IL-8 among many others. VEGF, which is a strong promoter of angiogenesis and neovascularization, is important for EPC cell differentiation, migration, proliferation and vascular remodeling, as can be concluded from previous studies [7,37]. SDF-1α, together with its receptor CXCR4, is crucial for BM-EPC mobilization from the bone marrow to the peripheral blood circulation and is important in cardiogenesis and vasculogenesis [7,36,38]. EGF plays a vital role in cell proliferation and differentiation by binding with EGF receptors. In the past few decades, it has been reported that EPCs provide protection by paracrine mechanisms involving release of a wide array of cytokines. In the present study, we have found that many important cytokines for regulating cell proliferation, survival, and the angiogenic process were clearly detected in the supernatants of BM-EPC cell sheets after 7 days of cultivation.
There are, however, still some problems to overcome for clinical application and some limitations of this study. One of the limitations is that the results presented in this study were obtained from in vitro experiments but not animal models. Another limitation is that the results of our study showed that the SDF-1α/CXCR4 axis may promote the secretion of paracrine factors; however, multiple mechanisms may involved in the paracrine action and the crosstalk of these paracrine factors was still unknown. Indeed, more detailed and speci c signaling research should be performed in future trials.

Conclusions
We successfully obtained BM-EPC cell sheets and con rmed that the sheets have superior proliferation and tube formation activity compared to those of EPC single-cell suspensions in vitro. The detailed research results showed that the SDF-1α/CXCR4 axis may promote the secretion of the paracrine factors VEGF, EGF and SDF-1α.

Declarations
Ethics approval and cinsent to participate: This study was reviewed and approved by the Animal Ethics Committee of Tianjin First Central Hospital (Number:E2017087L).

Consent for publication:
Not applicable Availability of data and materials: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing Interests
The Authors declare that they have no con ict of interests  Table   Table 1 Primer pairs used for reverse transcription -quantitative PCR