Novel Strategy for Isolation of Mice Bone Marrow – Derived Endothelial Cells (BMECs)

Background: In the bone marrow microenvironment, endothelial cells (ECs) are individual cells that form part of the sinusoidal blood vessels called "bone marrow endothelial niche." They account for less than two percent of the bone marrow cells. They may play critical functions in generating growth and inhibitory factors that promote the recovery of the hematopoietic stem cells. Methods: Two steps approach for isolation of bone marrow endothelial cells from mice. In brief, the bone marrow extracted from the mice long bones and culture overnight with DMEM supplemented with 20% FBS and antibiotics. The oating cells discarded, and the adhered section detaches with accutase and bone marrow endothelial cells selected using CD31 microbeads. The isolated bone marrow endothelial cells were cultured in dish pre-coated with rat-tail collagen type 1 with endothelial growth factor media. The cells were veried by confocal microscopy for morphology and tube formation by matrigel assay. We validate the purity of the cells by ow cytometry, RT-qPCR, immunouorescence staining, and immunoblotting. Finally, we induced the bone marrow endothelial cells with recombinant tumor necrosis factor-alpha. Results: Our ndings prove that the cell isolated are characteristic of bone marrow endothelial cells and response to tumor necrosis factor-alpha by an increase in proliferation and enhance the expression of adhesion molecules. Conclusions: This method simplies the extraction of primary bone marrow endothelial cells for in vitro studies. The signicance of this method will provide an excellent opportunity for stem cell research of endothelial cells function and dysregulation in vitro studies of mice model.


Introduction
The endothelial cells are part of the sinusoid-vascular niche in the bone marrow microenvironment. They play a signi cant role in producing growth and inhibitory cytokine that regulates the function of the hematopoietic stem cells. Also, they display adhesion molecules that interconnecting the hematopoietic progenitor cells. In the bone marrow microenvironment, they line the lumen of the sinusoidal-vascular niche. In response to in ammatory stimuli, they increase in proliferation to maintain the integrity of the blood vessels. The in ammatory response releases cytokines that promote their activation, such as tumor necrosis factor, interleukin 6. These cytokines, in turn, increase the expression of the endothelial adhesion molecule, such as; E-selectin, Vascular adhesion molecules (VCAM − 1), and intracellular adhesion molecules (ICAM-1) respectively. (1)(2)(3). Recent studies have reported that the endothelial niche is divided into the sinusoidal place and vascular niche. They are both recognized by the positive marker for vascular endothelial adhesion molecule (VE-cadherin) and CD31, but the sinusoidal slot expresses positive attributes for Vascular endothelial growth receptor factor 3 (VEGFR-3)(4-7). Besides, most studies have documented that endothelial cells express a positive marker such as; von Willebrand factor (VWF), intracellular adhesion molecule 1 (ICAM-1 /CD106), vascular adhesion molecule 1 (VCAM-1/CD105), E-selectin, BMA120, and endothelial selective adhesion molecule (ESAM). (8)(9)(10).Endothelial cells dysfunction within the bone marrow environment due to chemotherapeutic agents or radiotherapy may result in the deletion of VEGFR2 in adult mice. It may prevent the renewal of the sinusoidal endothelial and the recovery of hematopoietic stem cells. (11). The isolation of endothelial cells from the bone marrow could use to evaluate the functions and malfunctions of the endothelial cell in vitro studies. There are different types of endothelial cells, such as human umbilical vein endothelial cells (HUVECs), and mouse brain-derived endothelial cells (MBDECs). The existence of endothelial cells in different locations exhibits similar functions, such as recruitment of progenitor cells in response to in ammatory stimuli and capillary-like lumen tube formation (12). Although one of the recommended methods to evaluate endothelial cells is by ow cytometry analysis. Still, the conviction of real endothelial cells has proven di cult due to the lack of a particular monoclonal antibody against the cells. In this study, we developed a strategic method for isolation of mouse bone marrow endothelial cells by cell adhesion method followed by single-cell suspension with magnetic MLECs beads incubation and bounded cell isolation. This protocol can use to evaluate the function and dysfunction of bone marrow-derived endothelial cells in vitro studies. We will also highlight factors that need to take into consideration when using this method.  Step-by step process of bone marrow extraction:

MATERIALS
1. Mice aged 8-12 weeks terminated by cervical dislocation, and the whole mice soaked in 70% ethanol for 2-5minutes, then placed on the sterile dissecting board on the laminar ow hook. The long bones of the femora and tibias pulled off with micro-dissecting scissors placed in sterile Dulbecco's phosphate buffer solution (DPBS).
2. The muscles were detached from the bones by forceps, and the bones scrubbed to remove any residual soft tissues. The bones were washed twice with DPBS solution containing 1mmEDTA. One edge of the long bones cut -off place in a new dish containing the DPBS/EDTA solution.
3. An 18G needle pushed to the bottom of the 0.5 ml nest microcentrifuge tube. The cut edge of the long bone inverted downwards in the 0.5 ml nest microcentrifuge (maximum of 2 tibias and 2 femora) and the lid closed.
4. The nest of the 0.5 ml micro-centrifuge tube transfer into a 1.5 ml centrifuge tube, sealed with Para lm and centrifuge at 15,000 g for 30 seconds. The nest 0.5 ml microcentrifuge discarded and the visible pellet at the bottom of the 1.5 ml Eppendorf tubes suspended with sterile DPBS/EDTA solution.
5. The suspended bone marrow cells with the PBS/EDTA solution, ltered with 70um cell strainer (Biologix group limited) and centrifuge at 300 g for 5 minutes at room temperature. The pellet resuspended in DMEM supplemented with 20% fetal bovine serum (FBS), and 500 ul of penicillin (10,000uints/ml) /streptomycin 10,000ug/ml. 6. The cells plated in a sterile 10 mm dish with a minimum of 10 9 cells/plate and incubated in a 5% CO 2 humidi ed incubator overnight.

DAY 2
Step-by-step Process of isolating bone marrow-derived endothelial cells: 1. After overnight incubation, the oating cells poured off. The adhered cells were washed with prewarmed DPBS twice and detached with appropriate accutase solution for 15 minutes at room temperature.
2. The detachment stopped by adding the harvesting buffer PBS, 2% FBS, 1 mm Ethylenediaminetetraacetic acid (EDTA), and penicillin/streptomycin) with repeated pipetting to detached the remaining cells. The detach cells transferred into 15 ml tubes, centrifuge at 300 g for 10 minutes at room temperature. The total cell number determined by hemocytometer or automated cell counting machine.
3. The pellet cells resuspended with the harvesting buffer solution CD31 microbeads by manufacturer's protocols and stored at 4 0 C for 15 minutes.
4. The cells resuspended with 10 ml of harvesting buffer solution, then centrifuge at 300 g for 10 minutes at room temperature to wash off the excess beads from the cells after 15 minutes incubation.
5. The MS column attached to the magnetic washed once with the harvesting buffer, bone marrow cells pass through the column. The magnetic cells were washed three times with the harvesting buffer followed by a single wash with endothelial cell medium containing the factors.
6. The magnetic cells pushed into new sterile tubes with the endothelial cell media containing the additional growth factors, the total cells count determined by hemocytometer or automated cell counting machine.
7. The plate pre-coated with rat-tail collagen type 1, wash with sterile DPBS solution twice followed one wash with the endothelial cells growth media. Appropriate cell number seeded into the coated dishes and incubate into the incubator.

CHARACTERIZATION OF PRIMARY BONE MARROW-DERIVED ENDOTHELIAL CELLS (BMDECS).
Bone endothelial cell structure visualization The cultured cells sequentially observed every day for the capillary-like structure appearance of endothelial cells with confocal microscopy and photographed. (Nikon Eclipse Ti microscopy, Tokyo, Japan).

Matrigel capillary tube formation assay of BMDECs
Matrigel was allowed to thaw on ice overnight according to the manufacture's protocols. at room temperature (x2), incubated with the recommended dilution of antibodies, store at 4 0 C for one hour, and analyzed by ow cytometry (BD LSRFortessa ™) within 24 hours. The lower threshold uses to exclude debris and the live cells with gating (20,000 cells) according to FSC x SSC, followed by sections containing the antibodies. The data retrieve from the ow cytometry software and analyze by ow Jo software version 7.6.2.
Characterization of primary bone marrow-derived endothelial cells by Real-time quantitative PCR (RT-qPCR) To verify the molecular expression of the bone marrow endothelial cells, total RNA extracted from the cells after 7 days of incubation and the negative cells immediately after isolation using Trizol reagents (TIANGEN Cat#dp424). The cDNA is synthesized by using 5X All in one RT Master mix (Cat.No.G492) and kept at -20 0 C. Primers sequences and probe are shown in (Table 1). For RT-qPCR, the synthesized cDNA samples 10 ng were ampli ed with the SYBR green master mix in a nal volume of 20 ul, as described in our previously published article (13). The mean threshold values are used to evaluate the molecular gene expression with normalization with mouse beta-actin.

Validation of bone marrow endothelial cells by immunoblotting analysis
To examine the molecular expression of bone marrow endothelial cells. The cells were placed in a plate pre-coated with rat tail collagen type 1 washed with cold PBS, and a herpes-chap lysis buffer containing the protease inhibitors pour into the dish, incubate for 10 minutes, and adherent cells scraped off with cell scraper. The lysed cells were centrifuged at 20,000 g for 30 minutes at 4 0 C. While For the non-endothelial cells (CD31 negative), the protein isolated immediately after isolation and protein stored at -20 0 C until ready for use. The cell supernatants run on SDS-PAGE 8-12% gel (BIO-RAD; Hercules, CA). The proteins were transferred to the P 0.45 PVDF blotting membrane (Amersham™Hybond™ Germany) by the wet transfer method. Primary and secondary antibodies are shown in ( Table 2).

Characterization of BMDECs by immuno uorescence staining
To certify the bone marrow endothelial cells for the expression of CD31 (PECAM-1), VE-cadherin (CD144), and ICAM-1 with passage zero determine by direct immuno uorescence staining. The CD31 positive cells were plated into pre-coated 48-well with rat tail collagen type 1 for 5-7 days, as described above. After 60-70% of the con uence, the medium removed, cells xed with 4% paraformaldehyde (PF for 20 minutes at RT, followed by two wash 3 minutes apart with PBS. The cells were permeabilized with 100% cold methanol at room temperature for 20 minutes, rinse with PBS three times and blocked with 1% BSA/PBS for 1 hour at room temperature. Cells incubated with the recommended dilution of primary antibodies overnight at 4 0 C. The cells were cleaned twice with PBS and counterstained with Hoechst for 5 minutes at 37 0 C. The cells were washed with PBS and imaging acquired using inverted Nikon microscopy (Nikon Eclipse Ti microscopy, Tokyo, Japan). Primary and secondary antibodies are shown in (Table 2).

INDUCTION OF PRIMARY BONE MARROW DERIVED ENDOTHELIAL CELLS BY RECOMBINANT TUMOR
NECROSIS FACTOR ALPHA (TNF-α).

Assessment of primary bone marrow-derived endothelial cell proliferation
To examine the cell proliferation of primary bone marrow-derived endothelial cells, 10 5 cells were seed in 48 well plates incubated for 7 days. After 7 days, the BMDECs was stimulating with recombinant TNF-α (10 ng/ml) and the control with 1% FBS with DPBS for 48hrs, the media containing the recombinant cytokine and FBS/DPBS were replaced with new endothelial media and incubated for another 7 days. The cells were harvest by trypsin/ETDA ( VICMED 0.25% 0.02%), centrifuge at 350 g for 5 minutes, wash 2x with PBS, and xed with 70% cold -ethanol at -20 0 C for 1hour. The xed cells centrifuge as above followed by washed with FACS buffer incubated with Ki-67 (FITC, anti-mouse, Biolegend®) and F4/80 (APC, anti-mouse as isotype control Biolegend®) for 30 minutes at room temperature. The data acquired by ow cytometry. For the cell count, the cells were stain with trypan blue and live cells counted by hemocytometer.

The initiation of bone marrow endothelial cells by recombinant tumor necrosis factor-alpha
The molecular expression of markers speci c for bone marrow endothelial cells veri es by; real-time quantitative polymerase chain reaction (RT-qPCR) or immunoblotting and immuno uorescence staining.
The cells were cultured for 7 days to form a con uence, then stimulated with TNF-A at (10 ng/ml) and vehicle control (2% fetal bovine serum in PBS) for 48hrs. Cells were then harvested for RT-qPCR, western blotting analysis and stained for immuno uorescence, all samples done in triplicate, and results expressed in mean with standard deviation.

Statistical analysis
All data were statistically analyzed using Graph Prism 6 and paired two-tailed student's test used for comparison mean ± standard deviation. P-values ≤ 0.05 are considered statistically signi cant.

Results
Morphological observation and tube formation of bone marrow endothelial cells: In brief, after isolation of the bone marrow endothelial cells, as shown in Fig. 1 Real-time quantitative PCR and immunoblotting: The fold change of the relative messenger RNA for bone marrow endothelial cells is more signi cant than the non-endothelial cells for ICAM-1, VCAM-1 ESAM, and VE-cadherin, respectively, as shown in gure (4A-D). Furthermore, the immunoblotting shows an increase in the expression of E-selectin, VE-cadherin, PECAM-1, ICAM-1, and VCAM-1, respectively, as shown in Fig. 4E.
Certi cation of primary bone marrow-derived endothelial cells by immuno uorescence staining: To certi ed the purity of the primary bone marrow-derived endothelial cells, the cells were cultured in a 48well plate, after 60-70% of the con uence is achieve then tested against anti-CD31 (PECAM-1), anti-VEcadherin (CD144) and anti-CD106 (ICAM-1) respectively. The cell shows the expression of three EC markers; PECAM-1, VE-cadherin, and ICAM-1, respectively, as shown in Fig. 5 (A-C). The mean uorescence was quanti ed using Image J software on three experimental repeats, which reveals that expression of the EC-markers in order PECAM-1 > VE-cadherin > ICAM-1 as shown in Fig. 5(D).
The response of bone marrow endothelial cells to tumor necrosis factor-alpha: Brie y, the bone marrow endothelial cells culture for seven days and treated with recombinant tumor necrosis factor-alpha (10 ng/ml) for 48 hours and untreated cells with 2% FBS in PBS solution. Delta-delta threshold values determined the relative mRNA expression. The bone marrow endothelial cells response to tumor necrosis factor-alpha with the increase in fold change of; ICAM-1 (110.6 ± 19.8) and VCAM-1 (3.972 ± 0.6093), VEcadherin (11.46 ± 2.034), and ESAM (3.556 ± 0.4622) respectively as shown in Fig. 6(A-D p < 0.05) versus the control. The immunoblotting results also con rmed an increase in protein expression of ICAM-1 and VCAM-1, respectively, as shown in Fig. 6 (E-J, P < 0.05). However, the protein expression of the VEcadherin molecule decrease in response to TNF-α. The immuno uorescence staining also con rmed an increase in protein expression of ICAM-1 and decrease protein expression of VE-cadherin compared to the control, as shown in Fig. 7 (C-E, P < 0.05).Also, the bone marrow endothelial cells respond to TNF-α by increases in number, as shown in Fig. 7(A-B p < 0.05). This data indicates that TN-α enhances the proliferation and growth of bone marrow endothelial cells in vitro studies.

Discussion
In this study, we outline the step-by-step process of extracting bone marrow endothelial cells from mice long bones. Since bone marrow, endothelial cells are a critical component of the sinusoidal-vascular niche in the bone marrow microenvironment. There are no established cell lines for in vitro studies of bone marrow endothelial cells, which respond to in ammatory stimuli and promote hematopoietic stem cell regeneration. The morphological identi cation of the endothelial cells is a critical step in the process of demonstrating a functional vascular network that is associate with proliferation and differentiation, followed by elongation and assemble to a capillary-like a lumen/linear cord-like vessels forming an infusible vascular tube. (14)(15)(16) The amboidal -like the shape of the primary bone marrow-derived endothelial cells seen on the third-day post-culture, atypical cobblestone structure of the cells appeared on day 7, and the perfusable capillary-like lumen forming tube con rmed with Matrigel assay. The bone marrow microenvironment consists of three vascular networks; arterioles, transitional, and sinusoids. The endothelial cells are enclosed to sinusoid-vascular that form the endothelial -vascular niche in the hematopoietic stem cell that supports the maintenance and retaining the function of the stem cell niches. Besides, within the perivascular slots of the HSC they are interconnected by endothelial adherent molecules, including; VE-cadherin and ESAM as well as endothelial immunoglobulin-like adhesion molecules including; PECAM-1 (CD31), E-selectin, ICAM-1 (CD106) and VCAM-1 (CD105) (5,9,17,18). Therefore, validation of primary bone marrow-derived endothelial cells using these molecules will be essential in the isolation of pure endothelial cells from mice bone marrow. The cells isolated demonstrate the characteristic expression of these adhesion molecules. We further veri ed by the expression of the relative messenger RNA and the protein expression of these adhesion molecules, respectively. Also, primary bone marrow-derived endothelial cells were certi ed by immuno uorescence staining and show features of these adhesion molecules in sequential order PECAM-1, VE-cadherin and ICAM-1 respectively. Finally, we evaluated the response of bone marrow endothelial cells with recombinant tumor necrosis factor-alpha. Our results show that bone marrow endothelial cells increase in proliferation and expression of adhesion molecules, respectively. Also, the protein expression of vascular endothelial cadherin decrease with the change in the typical shape of the cells (19-22).
The importance and future application of bone marrow endothelial cells: The method can be used in vitro study to evaluate the functions and dysregulation of bone marrow endothelial cells for stem cell research of mice model. The signi cance of this method will provide an excellent opportunity to evaluate the endothelial-vascular niche in the hematopoietic stem. This technique will improve the practicality of endothelial stem cell research.

Conclusion
We established a method for isolation of primary bone marrow-derived endothelial cells from mice bone marrow, which can be used to evaluate the function of endothelial cells in vitro studies. We suggest the following factors must be considered when isolating these cells.

PRE-REQUISITES PROCESSING
1. Before isolation, boil all instruments, air dry in the oven for at least 30 minutes, and add Gauze to autoclave.
2. Prepare fresh DPBS solution with PH 7.2-7.6, 0.5 molar EDTA and sterilize 3. Sterilize 0.5 ml tube and 1.5 ml tubes for density centrifugation of the bone marrow cell from the mice bones.
4. Prepare sterile 1mmEDTA/DPBS and harvesting buffer solution kept at 4 0 C for 30 days only.
5. Pre-coat the dish with rat tail collagen overnight at 37 0 C in the incubator, wash three times with sterile DPBS, air dry for 30 seconds before pouring the endothelial cell growth media ISOLATION 1. Try to spend less time on harvesting the bones from the mice, wash bones with sterile DPBS, and a nal wash with DPBS containing 1mmEDTA to prevent blood clotting and clumping of the cells. 2. Use a 5 ml syringe needle to perforate the center of the 0.5 ml tube and invert the cut edge of the bones (maximum 4 2 tibias, 2 femora), suspend the pellet with DPBS/1mmEDTA.
3. Incubate the bone marrow cells, not more 24 hours. 4. Change medium after 3-4 days to maintain the cell numbers.

LIMITATION
1. To get enough cells its involve a large number of mice (age 6-12 weeks), especially for immunoblotting techniques.
2. The cell passage number should not exceed two passages (Passage 0 and 1 recommended). More than 3 passages, the cells are prone to fungal infection and cell drift. 3. Contamination of primary bone marrow-derived endothelial cells is common with macrophages, broblasts, if incubated cells for than 24 hr. To avoid that, try to harvest cells less than 24 ours incubation and repeats passage for the cells twice via the column.

The endothelial growth media is susceptible to infection, have an excellent working sterile
environment key to get optimal results. Density centrifugation minimizes the risk of contamination. 5. The primary bone marrow-derived endothelial have di culties attached to the glass slide for immuno uorescence staining for this procedure using 48 or 96 wells to get optimal results.

Data availability
All relevant data in the study included in the article; further inquiries can be directed to the corresponding authors.

Con ict of interest
The authors declared that this study was not conducted for nancial gain or any commercial part that could be construed as a potential con ict of interest   Figure 1 Step