​Chip Detection of Rabbit Peripheral Blood Mononuclear Cells and 293T Cells Induced by Chicken Egg White Extract Shows Significant Differences in the Signaling Pathways that Regulate the Pluripotency of Stem Cells

To date, many studies have reported the composition and function of chicken egg white extract (Fang et al., 2012; Kodama et al. 2012; Abeyrathne et al. 2014). However, few studies have detected whether chicken egg white extract can induce reprogramming of somatic cells (Mizutani et al. 2012; Qiu et al. 2013; Yang et al. 2013; Ruan et al. 2014; Ruan et al. 2017). Previous studies have shown that human cells can be directly reprogrammed into an induced pluripotent stem cell (iPSC) state by expressing low levels of transcription factors (Takahashi et al. 2007; Yu et al. 2007). There are also reports that animal egg cell extracts can reprogram somatic cells, including pig egg extract (Miyamoto et al. 2009) [11] and toad egg (Hansis et al. 2004; Miyamoto et al. 2007) extracts can reprogram somatic cell nuclei. In this study, we used 40% chicken egg white extract to induce rabbit peripheral blood mononuclear cells and 293T cells, cocultured them for 3 days, assessed the uninduced and induced cells with a stem cell protein chip and found that in the rabbit peripheral blood mononuclear cells treated with chicken egg white extract, 10 stem cell-related proteins were significantly increased. After 293T cells were induced with chicken egg white extract, 4 stem cell-related proteins were also significantly increased. The results of KEGG enrichment analysis showed that before and after induction of rabbit peripheral blood mononuclear cells, signaling pathways are involved in regulating the pluripotency of stem cells. Before and after the induction of 293T cells, signaling pathways are involved in regulating the pluripotency of stem cells. This finding shows that the cell has undergone reprogramming and it is reported as follows. We used advanced chip technology to detect the effect of chicken egg white extract to reprogram somatic cells and confirmed its effect. This research is very innovative.
 

Preparation of chicken egg white extract
 
Approximately 20 ml of egg white was aspirated from the egg, 20 ml of PBS was added, mixed well, frozen and thawed 3 times and centrifuged and the supernatant was collected for later use.
 
This work was carried out at The Basic Medical Laboratory of 920th Hospital of Joint Logistics Support Force of PLA in 2021.
 
Isolation and culture of rabbit peripheral blood mononuclear cells (R-PBMCs)
 
First, 25 ml of blood was collected from the rabbit ear venous vein, 12.5 ml of lymphocyte separation liquid was added to a centrifuge tube, blood was carefully added on top of the separation liquid and the sample was centrifuge at 2000 rpm for 20 min. Then, the buffy coat layer was aspired and the cells were washed twice. Two culture bottles were used: one had ordinary culture medium and the other had ordinary culture medium containing 40% chicken egg white extract. The ordinary culture medium was DMEM-F12 medium supplied with 10% fetal bovine serum. Three days after induction, the cells were collected to generate the stem cell protein chip. The 293T cell strain was purchased from the cell bank of the Kunming Institute of Zoology, Chinese Academy of Sciences. Two bottles of 293T cells were also cultured. One bottle had normal medium and the other had normal medium containing 40% chicken egg white extract. After 3 days of induction, the cells were collected. The stem cell protein chips were used to detect Stem Cell Associated Proteins in Cells. After the cells were collected and centrifuged, the supernatant was discarded and the pellet was quickly frozen in a refrigerator at -80°C.
 
Sample preparation
 
The lysate (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS) was added to the cell pellet and mix repeatedly and centrifuged at 14000 rpm for 10 min in a refrigerated centrifuge; the supernatant was collected.
 
Protein concentration determination and dilution scheme
 
After the cells were lysed, the protein concentration was determined (BCA method, Pierce Company, catalog number: 23227). A BCA kit was used to detect the protein concentration of the tissue lysates. The measurement results and sample preparation are shown in Table 1.


 
Determination
 
Complete drying of the slide chip
 
The slide chip was taken out of the box (RayBio® Human Stem Cell Array G-Series 1). After equilibration at room temperature for 20-30 minutes, the packaging bag was opened, the sealing strip was uncovered and then, the chip was placed in a vacuum desiccator or dried at room temperature for 1-2 hours.
 
Blocking and incubation
 
1. First, 100 µL of 1x blocking solution (1% BSA in TBST) was added to each chip hole and incubate for 1 hour on a shaker at room temperature to avoid air bubbles.
2. The blocking solution was removed, 100 µL of sample was added to each well and one sample per array was incubated overnight at 4°C with shaking (load at 500 µg/ml).
3. A Thermo Scientific Wellwash Versa chip washer was used to clean the slides in two steps. First, the cells were washed with 1x Washing Solution I; 250 mL of 1x Washing Solution I was added per well and 10 washes were performed, with shaking for 10 s each time at a high intensity, followed by dilution with 20x solution I with deionized water. Then, 1x Washing Solution II was added to the channel for cleaning. A total of 250 μL of 1x Washing Solution II per well was used for 6 washes, with shaking for 10 s each time. The shaking intensity was high and 20x Washing Solution II was diluted with deionized water.
4. Biotin-labeled antibody was prepared, the biotin-labeled antibody tube was rapidly centrifuged and 300 µL of 1x blocking solution was added to each tube and mixed well. Then, 70 μL of biotin-labeled antibody was added to each well and incubated for 2 h at room temperature.
5. Cleaning was performed, as described in step 3.
6. Then, 70 μL of 1500 times diluted HiLyte Plus^TM 555 streptavidin-Fluor was added to each well (after rapid centrifugation, 1.5 mL of blocking solution was added to the fluorescent agent-streptavidin tube) and the glass slide was sealed with a sealing strip. Then, the slide was wrapped with aluminum foil and incubated for 1-2 hours at room temperature with shaking at room temperature in the dark.
7. Cleaning was performed, as described in step 3.
 
Fluorescence detection was performed
 
1) The frame of the slide was removed, taking care not to touch the side of the slide with printed antibodies.
2) A laser scanner such as InnoScan 300 was used to scan the signal using the Cy3 or green channel (excitation frequency = 532 nm).
(i) Instrument model: InnoScan 300 Microarray Scanner.
(ii) Manufacturer: Innopsys.
(iii) Place of origin: Parc d’Activités Activestre; 31 390 Carbonne - France.
(iv) Scanning parameters: wavelength: 532 nm; resolution: 10 µm.
Chip analysis software was used to extract data and AAH-SC-G1 data analysis software was used for data analysis. The original data obtained from the chip scanning were processed by Raybiotech software for chip background removal, normalization between chips and differential protein screening.

1. After rabbit peripheral blood mononuclear cells were induced with chicken egg white extract, 10 stem cell-related proteins were found to be significantly increased (Table 2).



2. After 293T cells were induced with chicken egg white extract, there were also 4 stem cell-related proteins that were significantly increased (Table 3).



3. The results of KEGG enrichment analysis showed that before and after induction of rabbit peripheral blood mononuclear cells, signaling pathways were involved in regulating stem cell pluripotency (Fig 1).



4. The results of KEGG enrichment analysis showed that before and after induction of 293T cells, signaling pathways were involved in regulating stem cell pluripotency (Fig 2).



AFP, BMPR-1A, CD38, E-cadherin, HCG-beta, NANOG, NESTIN, Brachyury, GATA4, SOX2, SOX17 and VEGFR2 are proteins expressed in stem cells. When the cells are transformed into pluripotent stem cells, the expression of these proteins significantly increases (Teshigawara et al. 2017; Wang et al. 2017). Thus, when the expression of these proteins is significantly increased in the cell, the cell may be transformed into a pluripotent stem cell.

We found that the expression of 10 stem cell-related proteins in rabbit peripheral blood mononuclear cells cocultured with 40% chicken egg white extract for 3 days was significantly increased. KEGG enrichment analysis of the rabbit peripheral blood mononuclear cells was performed before and after induction. The signaling pathway was related to regulation of the pluripotency of stem cells. In 293T cells cocultured with 40% chicken egg white extract for 3 days, the expression of 4 stem cell-related proteins was significantly increased. The results of KEGG enrichment analysis showed that before and after induction of 293T cells, signaling pathways were involved in regulating stem cell pluripotency. This finding further proves that the chicken egg white extract promotes somatic cell reprogramming.

Compared with pig egg extract and toad egg extract, chicken egg white extract is more convenient to obtain, the amount obtained is greater and the extraction process should be sterile (Monika 2021). This extract can be directly used for cell culture without filtration and sterilization and has greater application value (Lyayla 2021). In our previous research, we found that chicken egg white extract can promote cell proliferation and increase the expression of the pluripotent factor NANOG. In this research, we further used a stem cell protein chip to detect the proteins expressed by a variety of stem cells (Bujun 2021). The results showed that in the rabbit periphery, the expression of 10 stem cell-related proteins (Jyotsana  2021) in blood mononuclear cells was significantly increased and the expression of 4 stem cell-related proteins in 293T cells was significantly increased, indicating that the chicken egg white extract contains components that promote somatic cell reprogramming. We speculate the components of chicken egg white extract may have contributed to the regulation of pluripotency of stem cell are proteins with molecular weight less than 10KDa. The proteins effect or the function of those proteins may be promote somatic cell reprogramming. Further research on chicken eggs and the active ingredients in the clear extract is needed. This extract will have a wide range of applications in cell biology (Liyun  2021). The increase of stem cell protein expression in cells after induction with 40% final concentration of chicken egg white extract indicates the transformation of somatic cells to stem cells, indicating that there are small protein molecules in the chicken egg white extract that can induce reprogramming, which will help the research in the field of cell biology.

Cocultivation with 40% chicken egg white extract can induce reverse differentiation of cells into stem cells and the expression of stem cell-related proteins is significantly increased.

We thank American Journal Experts for assisting with the English preparation of this manuscript. 

Author contribution
 
Jie He, Ping Mo, Guang-ping Ruan and Xiang Yao made substantial contributions to the study conception and design, data acquisition and data analysis and interpretation. Jie He, Guang-ping Ruan, Zai-ling Yang, Kai Wang and Xiang Yao conducted the experiments. Xiang Yao and Jie He agree to be accountable for all aspects of the work and ensure that questions related to the accuracy or integrity of any part of the study will be appropriately investigated and resolved. Xing-hua Pan and Guang-ping Ruan provided final approval of this version of the manuscript for publication. Guang-ping Ruan, Xing-hua Pan, Jie He, Xiang-qing Zhu and Rong-qing Pang were involved in drafting the manuscript or revising it critically for important intellectual content. All authors read and approved the final manuscript.
 
Funding statement
 
This work was supported by grants from the Yunnan Science and Technology Plan Project Major Science and Technology Project (2018ZF007), the Yunnan Province Applied Basic Research Program Key Project (2018FA041, 202101AS0 70039), the General Project of Yunnan Applied Basic Research Program (202101AT070212), the National Natural Science Foundation (31970515) and the 920th Hospital of the PLA Joint Logistics Support Force In-hospital Technology Plan (2019YGB17, 2019YGA05).
 

All authors declare that they have no conflict of interest.