Methods to stimulate cell growth

Abstract

This provides a method for stimulating cell growth. [Solution] A method for stimulating cell growth, comprising the step of bringing cells into contact with a composition containing isolated lens epithelial cell-derived growth factor (LEDGF).

Description

Technical Field

[0001]

[0001] The present invention relates to a method for stimulating cell growth, particularly a method for stimulating cell growth with lens epidermal dermal growth factor (LEDGF).

Background Art

[0002]

[0002] Fetal serum contains factors that promote changes in gene expression and enable the infinite culture of cell lines such as RAW 264.7 mouse macrophages. Insulin, nerve growth factor, epidermal growth factor, insulin-like growth factor, fibroblast growth factor (FGF), platelet-derived growth factor, and transforming growth factor (TGF) have been shown to control cell proliferation. However, the effect of growth hormone is inferior to that of fetal serum even after supplementation with albumin, transferrin, amino acids, or diluted serum (Yao, T and Y. Asayama, Animal-cell culture media: History, characteristics, and current issues. Reprod Med Biol, 2017.16(2): p. 99-117), and therefore, all the factors enabling infinite cell growth have not yet been determined (Kwon, D. et al., The Effect of Fetal Bovine Serum (FBS) on Efficacy of Cellular Reprogramming for Induced Pluripotent Stem Cell (iPSC) Generation. Cell Transplant, 2016.25(6): p. 1025-42). Fetal serum or platelet lysates have also been shown to grow clonal cell lines, which may be applicable to therapy and have fundamental biochemical importance (Castellano, J.M. et al., Human umbilical cord ​​Plasma proteins revitalize hippocampal function in aged mice. Nature, 2017. 544(7651): p.488~492. There have been considerable efforts to create cell growth media containing the type of growth factor found in natural serum used in cell culture experiments, and attempts to enumerate fetal serum proteins from umbilical cords or other approaches (Li, C., F. Wang, and L. Liu, Ultra-high performance liquid chromatography-mass spectrometry for analysis of newborn and fetal bovine serum components. Nan Fang Yi Ke Da Xue Xue Bao, 2014. 34(5): p.751~3). [Overview of the Initiative] [Means for solving the problem]

[0003]

[0003] Lens epithelial cell-derived growth factor was first identified in fetal serum and found to stimulate cell growth.

[0004] This disclosure provides a method for stimulating cell growth, comprising the step of contacting cells with a composition containing isolated lens epithelial cell-derived growth factors.

[0004]

[0005] In one embodiment of the present disclosure, the composition is purified fetal serum.

[0006] In one embodiment of the present disclosure, the composition further comprises a hormone.

[0007] In one embodiment of this disclosure, the hormone is a growth-related hormone.

[0005]

[0008] In one embodiment of this disclosure, the hormone is insulin, insulin-like growth factor 2, multiple EGF-like domain 11 (MEGF11), erythropoietin (EPO), Pigment epithelial cell-derived factor [PEDF, also known as serpin F1 (SERPINF1)], interleukin-17D (IL17D), interleukin-37 (IL37), interleukin-17C, interleukin-13, interleukin-2, retinol-binding protein 4 (RBP4), tissue factor pathway inhibitor (TFPI, preferably tissue factor pathway inhibitor 2), nerve growth factor beta polypeptide (NGFB), growth hormone somatotropin (GH1 / GH), multiple EGF-like domain 6 (MEGF6), tumor necrosis factor (TNF, preferably TNF alpha), cardiotrophin-like cytokine factor 1 (CLCF1), teratoma-derived growth factor (TDGF, preferably teratoma-derived growth factor-1), glycoprotein hormone subunit beta 5 (GPHB5) The following are selected from the group consisting of cytokine receptor-like factor 1 (CRLF1), transforming growth factor beta (TGFB, preferably transforming growth factor beta 2), growth hormone exon 5 (GHE5), CC motif chemokine ligand 21 (CCL21), platelet-derived growth factor (PDGF), platelet-derived growth factor receptor-like (PDGFRL), growth differentiation factor (GDF, preferably growth differentiation factors 15, 11, 6, 5, or 3), fibroblast growth factor (FGF, preferably fibroblast growth factors 18, 14, or 8), CXC motif chemokine ligand (CXCL, preferably CXC motif chemokine ligand 13, 9, or 6), and insulin-like growth factor-binding protein (IGFBP, preferably insulin-like growth factor-binding protein 4 or 2).

[0006]

[0009] In one embodiment of this disclosure, the cells are stem cells.

[0010] In one embodiment of this disclosure, the method is for stimulating more cell division cycles.

[0007]

[0011] In one embodiment of this disclosure, the method is for extending the lifespan of cells.

[0012] In one embodiment of this disclosure, the step of bringing the cells into contact takes place during cell culture.

[0008]

[0013] In one embodiment of this disclosure, the step of bringing the cells into contact is in situ.

[0014] This disclosure is described in detail in the following sections. Other features, purposes, and advantages of this disclosure can be found in the detailed description and claims. [Brief explanation of the drawing]

[0009] [Figure 1A]

[0015] Figures 1A-1C show the effects of fetal serum versus adult serum on the morphology of RAW 264.7 mouse macrophages cultured in DMEM + 5% fetal or adult bovine serum. Figure 1A, fetal bovine serum. [Figure 1B] Figures 1A-1C show the effect of fetal serum versus adult serum on the morphology of RAW 264.7 mouse macrophages cultured in DMEM + 5% fetal or adult bovine serum. Figure 1B shows adult bovine serum. [Figure 1C] Figures 1A–1C show the effect of fetal serum versus adult serum on the morphology of RAW 264.7 mouse macrophages cultured in DMEM + 5% fetal or adult bovine serum. Figure 1C, distribution of cell length in adult serum (ABS) versus fetal serum (FBS). Ten cells were measured from three spots on three coverslips from three independent batches of fetal serum versus adult serum. The longest axis of the cell was measured. F-statistic: 307.1 at 3 and 156 DF, p-value: <2.2e-16. [Figure 2]

[0016] This diagram shows flowcharts of the isolation and analysis steps for proteins (proteomics) and endogenous peptides (peptidomics). [Figure 3]

[0017] Figure 3A shows the fractionation of bovine plasma by a step gradient of NaCl in 20 mM Tris pH 8.85 buffer. Dumbroff protein assay. Figure 3B shows the fractionation of bovine plasma by a step gradient of NaCl in 20 mM Tris pH 8.85 buffer. Lanes: 1, molecular weight standard; 2, wash (W); 3, flow-through (FT). Quaternary amine (QA) fractions: 1, 0 mM; 2, 50 mM; 3, 100 mM; 4, 150 mM; 5, 175 mM; 6, 200 mM; 7, 225 mM; 8, 250 mM; 9, 300 mM; 10, 350 mM; 11, 400 mM; 12, 450 mM; 13, 500 mM; 14, 600 mM, 50% ACN; 16, 5% formic acid. [Figure 4]

[0018] Figure 4A shows the sequential extraction of low molecular weight polypeptides from serum using stepwise solubilization in organic / water. Dumbroff protein assay. Figure 4B shows the sequential extraction of low molecular weight polypeptides from serum using stepwise solubilization in organic / water. Lane: 1, molecular weight standard. Acetonitrile (ACN) / water fractions: 1, 90% ACN; 2, 70% ACN; 3, 60% ACN; 4, 50% ACN; 5, 40% ACN; 6, 30% ACN; 7, 20% ACN; 8, 10% ACN; 9, 5% ACN. [Figure 5]

[0019] Figure 5A shows the reproducibility of fetal serum samples versus adult serum samples. A QQ plot shows the normality of the log10 transformed intensity values. Figure 5B shows the reproducibility of fetal serum samples versus adult serum samples. Box plots show the mean log10 intensity, the variation in log10, and the 99% confidence interval for adult serum replicate versus fetal serum replicate. Processing:1, ABS QA rep1;2, ABS QA STYP rep1;3, ABS QA rep2;4, ABS QA STYP rep2;5, ABS QA rep3;6, QA ABS QA STYP rep3;7, ABS, ACN rep1;8, ABS ACN STYP rep1;9 ABS ACN rep2;10, ABS, ACN STYP rep2;11, ABS, ACN rep3;12, ABS ACN STYP rep3;13, FC QA rep1;14, FCS QA STYP rep1;15, FCS QA rep2;16, FCS QA STYP rep2;17, FCS QA rep3;18, FCS QA STYP rep3;19, FCS AACN rep1;20, FCS ACN STYP rep1;21, FCS ACN rep2;22, FCS ACN STYP rep2;23, FCS ACN rep3;24, FCS ACN STYP rep3. [Figure 6]

[0020] Figure 6A shows the total number of protein accessions from a bovine protein library corrected by the combined X! TANDEM and SEQUEST algorithms. Duplicate protein accessions. Figure 6B shows the total number of protein accessions from a bovine protein library corrected by the combined X! TANDEM and SEQUEST algorithms. Best correlation per gene symbol. [Figure 7]

[0021] Figure 7A shows the protein accessions identified by the SEQUEST algorithm. Figure 7B shows the protein accessions identified by the X! TANDEM algorithm. The bovine protein library contained 209,111 protein accessions. [Figure 8]

[0022] A figure showing the cumulative p - value and FDR - corrected q - value of non - overlapping peptides per gene symbol calculated from X! TANDEM results using the R statistical system. [Figure 9-1]

[0023] Figure 9A is a quantile plot showing the corrected difference (delta) in the observed frequencies of fetal serum vs adult serum. Delta of trypsin - digested peptide observed frequencies. Figure 9B is a quantile plot showing the chi - square value of the observed frequencies of fetal serum vs adult serum. Chi - square χ2 of trypsin - digested peptide. [Figure 9-2] Figure 9C is a quantile plot showing the corrected difference (delta) in the observed frequencies of fetal serum vs adult serum. Delta of trypsin - digested and / or STYP observed frequencies. Figure 9D is a quantile plot showing the chi - square value of the observed frequencies of fetal serum vs adult serum. Chi - square χ2 of trypsin - digested and / or STYP peptides. [Figure 10]

[0024] A figure showing the effect of insulin on the longest cell axis of RAW 264.7 macrophages in bovine adult serum (ABS) vs bovine fetal serum (FBS). The level of insulin added to each well of a 6 - well culture dish is shown. Letters indicate statistically significant differences by Tukey Kramer Honestly Significant Difference (HSD) test. [Figure 11]

[0025] A figure showing the effect of insulin on the longest cell axis of RAW 264.7 macrophages in bovine adult serum (ABS) vs bovine fetal serum (FBS). The level of insulin or lens epithelium - derived growth factor (LEDGF) added to each well of a 6 - well culture dish is shown. Letters indicate statistically significant differences by Tukey Kramer Honestly Significant Difference (HSD) test.

Mode for Carrying Out the Invention

[0010]

[0026] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings generally understood by those skilled in the art. The meaning and scope of such terms should be clear, but in the event of any potential ambiguity, the definitions provided herein shall prevail over any dictionary or external definitions.

[0011]

[0027] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to a plural object unless the context clearly indicates otherwise. Therefore, unless the context requires otherwise, a singular term shall include its plural form, and a plural term shall include its singular form.

[0012]

[0028] As used herein, the term "or" is used to mean "and / or" unless it is explicitly indicated that only one option is being referred to or that the options are mutually exclusive.

[0013]

[0029] This disclosure provides a method for stimulating cell growth, comprising the step of contacting cells with a composition containing isolated lens epithelial cell-derived growth factor (LEDGF).

[0030] Fetal serum contains factors such as alpha-fetoprotein (AFP), insulin, and insulin-like growth factor 2, which support the uninterrupted division and growth of cancer cell lines. However, not all of the peptides and proteins in fetal serum that regulate cell growth have been elucidated. In some embodiments of this disclosure, peptides from fetal serum versus adult serum were extracted in an organic solvent, and their proteins were degraded on a quaternary amine resin for trypsin digestion. The resulting peptides were analyzed by random and independent sampling using LC-ESI-MS / MS. Peptides and proteins were identified by the X! TANDEM and SEQUEST algorithms. Precursor and fragment intensity values ​​for peptide and protein assignments were collected in a SQL server for analysis in the R statistical system. 225,097 endogenous peptide sequences from peptidomics showed 23% overlap with 431,443 distinct peptide sequences from proteomics. However, independent sets of peptides showed 99.3% agreement in small sets of protein gene symbols, while most proteins were not observed. Consistent results from independent methods provided clear, unambiguous evidence that LC-ESI-MS / MS of plasma in a linear quadrupole ion trap exhibited a low error rate for peptide and protein identification. Fetal serum showed increased frequency of observed peptides derived from insulin and proteins involved in the AKT-HRAS pathway. Many growth factors, including lens epithelial cell-derived growth factor, were specific to fetal serum. Addition of insulin and / or LEDGF to adult serum partially restored the round phenotype of rapidly dividing cells, but not as effectively as in fetal serum. All peptides and proteins in the blood could be counted using a simple linear quadrupole ion trap, revealing all fetal serum-specific proteins that support the immortal growth of cells with an error of ≤1%. Therefore, one of the embodiment compositions was purified. It is fetal serum.

[0014]

[0031] One or more hormones may be added to the composition of the present invention. In one embodiment of the present disclosure, the hormones are growth-related hormones. Examples of hormones include insulin, insulin-like growth factor 2, multiple EGF-like domain 11 (MEGF11), erythropoietin (EPO), pigment epithelial cell-derived factor (PEDF), interleukin-17D (IL17D), interleukin-37 (IL37), interleukin-17C, interleukin-13, interleukin-2, retinol-binding protein 4 (RBP4), tissue factor pathway inhibitor (TFPI2), nerve growth factor beta polypeptide (NGFB), growth hormone somatotropin (GH1 / GH), multiple EGF-like domain 6 (MEGF6), tumor necrosis factor (TNF), and cardiotrophin-like hormones. Examples include, but are not limited to, cytokine factor 1 (CLCF1), teratoma-derived growth factor (TDGF), glycoprotein hormone subunit beta 5 (GPHB5), cytokine receptor-like factor 1 (CRLF1), transforming growth factor beta (TGFB), growth hormone exon 5 (GHE5), CC-motif chemokine ligand 21 (CCL21), platelet-derived growth factor (PDGF), platelet-derived growth factor derivative-like (PDGFRL), growth and differentiation factor (GDF), fibroblast growth factor (FGF), CXC-motif chemokine ligand (CXCL), and insulin-like growth factor-binding protein (IGFBP).

[0015]

[0032] After contacting cells with the composition of the present invention in vitro or in vivo, cell growth can be stimulated. Cell growth stimulated by the composition of the present invention includes producing more cell division cycles or extending the lifespan of cells. The in vitro contact step is carried out through a cell culture process.

[0016]

[0033] The following embodiments are provided to help those skilled in the art to put the present disclosure into practice. [Examples]

[0017] material and method material

[0034] The HPLC system was an Agilent 1100 (Santa Clara, CA, USA). The linear ion trap mass spectrometer was an LTQ XL (Thermo Electron Corporation, Waltham, MA, USA). The ceramic quaternary amine resin was from BioRad. C18 ZipTips were obtained from Millipore (Bedford, MA). The C18 HPLC resin was from Agilent (Zorbax 300 SB-C18 5 microns, 300 angstroms). The solvent was obtained from Caledon Laboratories (Georgetown, Ontario, Canada). Three independent batches of fetal bovine serum (FCS) and adult bovine serum (ABS) were supplied from Cell Grow, Sigma-Aldrich (Canada), Gibco by life technologies (New Zealand), MP Biomedical (MP, USA), Rocky Mountain Biologicals (USA), and Thermo Fisher. The samples were thawed, aliquoted, refrozen, thawed once before use, and the remainder discarded. Recombinant LEDGF (lens epithelial cell-derived growth factor) protein was supplied from MyBioSource (San Diego, USA). As described, the LEDGF recombinant protein was used in cultured mice (Mus) 0.1 mg of LEDGF, produced from musculus (mouse) cells, was resuspended in sterile PBS at a concentration of 0.1 mg / mL before use. Human recombinant insulin was supplied by Sigma-Aldrich (Canada). Human recombinant insulin was dissolved in sterile PBS at a concentration of 0.1 mg / mL before use. All other salts and Reagents were obtained from Sigma-Aldrich-Fluka (St. Louis, MO) unless otherwise specified.

[0018] Protein separation on quaternary amine resin

[0035] Proteins from 25 μL of serum were diluted in 200 μL of 20 mM tricine pH 8.8 binding buffer.

[0019]

[0036] A new disposable quaternary amine column was prepared for each protein sample (Tucholska, M. et al., Human serum proteins fractionated by preparative partition chromatography prior to LC-ESI-MS / MS. Journal of (Proteome Research, 2009.8: pp. 1143-1155). A 100 μL column of quaternary amine resin was inserted into a 1.5 mL injector inside a 15 mL tube (Marshall, J. et al., Human serum proteins preseparated by electrophoresis or chromatography followed by tandem mass spectrometry. J Proteome Res, 2004.3(3): pp. 364-382). The quaternary amine resin in 50% slurry was packed by cutting the valve to act as a buffer reservoir and packing the tip with glass wool frit (Tucholska, M. et al., Human serum proteins fractionated by preparative partition chromatography prior to LC-ESI-MS / MS. Journal of Proteome Research, 2009.8: pp. 1143-1155). The resin was equilibrated with binding buffer before introducing the sample by gravity. The resin was washed with 3 volumes of buffer before the increasing salt step gradient. To avoid cross-contamination, the quaternary amine column used for preparation was discarded after one use. The sample was then digested with 1 / 100 wt / wt trypsin for 12 hours, and reduced in 2 mM DTT at 50°C for 30 minutes before a second digestion with 1 / 100 wt / wt trypsin. The sample was quenched with 5% formic acid and dried.

[0020] Endogenous peptide extraction

[0037] A serum sample (200 μL) was precipitated in a 2 mL sample tube with 9 volumes of acetonitrile (90% ACN) (Tucholska, M. et al., Endogenous peptides from biophysical and biochemical Fractionation of serum was analyzed by matrix-assisted laser desorption / ionization and electrospray ionization hybrid quadrupole time-of-flight (Analytical Biochemistry, 2007, 370: pp. 228-245), followed by stepwise extraction of the pellet. Acetonitrile / water extracts were separated from the insoluble pellet in each step fraction by centrifugation at 12,000 RCF for 5 minutes. The organic precipitate (pellet) containing a very large total amount of endogenous polypeptide was manually resuspended in a step to increase the water content, yielding 10 fractions from 90% ACN to 10% ACN, followed by water, after which 5% formic acid was added (Dufresne, J. et al., A method for the extraction of the endogenous tryptic peptides (peptidome) from human EDTA plasma (Anal Biochem, 2018). The acetonitrile / aqueous phase containing the peptide was collected, transferred to a fresh sample tube, dried under vacuum in a rotary freeze-dryer, and stored at -80°C for subsequent analysis.

[0021] C18 chromatography for peptide preparation

[0038] C18 separation for preparation provided the best results for peptide and phosphopeptide analysis in a "blinded" comparison (Krokhin, OV, W. Ens, and KGStanding, MALDI QqTOF MS combined with off-line HPLC for characterization of protein primary structure and post-translational modifications. J Biomol Tech, 2005.16(4):p.429~40). Each fine fraction was collected using a new disposable C18 preparation "Zip Tip" column. Solid-phase extraction with C18 for LC-ESI-MS / MS was performed as previously described. The C18 chromatography resin (Zip Tip) was moistened with 65% acetonitrile before equilibration in water containing 5% formic acid. Plasma extracts were dissolved in 200 μL of 5% formic acid in water. The resin was washed with at least 5 volumes of the same binding buffer. The resin was eluted with ≥3 column volumes of 65% acetonitrile (2 μL) in 5% formic acid. To avoid cross-contamination, the C18 resin used for preparation was discarded after a single use.

[0022] AnalysisLC-ESI-MS / MS

[0039] To completely prevent even the slightest possibility of cross-contamination between serum samples, new disposable nanoanalytical HPLC columns and nanoemitters were fabricated to record each set of quaternary amine (proteomics) and organic (peptidomics) extractions ((n=3 fetuses + n=3 adults) samples × 2 fractionation methods = 12 columns). Each fetal or adult serum sample resulted in two sample fraction sets (10 peptide fractions and 16 protein fraction digests per sample = 26 fractions) for a total of 156 LC-ESI-MS / MS experiments (2 treatments × 3 replicates × 26 fractions). The ion trap was cleaned and tested for sensitivity to angiotensin and gluten-fibrinogen before recording (Dufresne, J. et al., Re-evaluation of the rabbit myosin protein standard used to create the empirical statistical model for decoy library searching. Anal Biochem, 2018, 560: pp. 39-49; Thavarajah, T. et al., Re-evaluation of the 18 non-human protein standards used to create the Empirical Statistical Model for Decoy Library Searching. Anal Biochem, 2020: pp. 113680).Each disposable C18 analytical column was conditioned and quality-controlled with a mixture of three non-human protein standards using digestion of bovine cytochrome C, yeast alcohol dehydrogenase (ADH), and rabbit glycogen phosphorylase B to confirm the system's sensitivity and mass accuracy (Bowden, P. et al., Quantitative statistical analysis of standard and human blood proteins from liquid chromatography, electrospray ionization, and tandem mass spectrometry. Journal of proteome research, 2012.11: pp. 2032-2047). The conditioned column was thoroughly washed in 50% acetonitrile before introducing the sample fraction set. Stepwise fractions were collected on a C18 preparation microcolumn, eluted in 2 μL of 65% ACN and 5% formic acid, diluted in 18 μL of 5% formic acid in water, immediately loaded manually into a 20 μL metal sample loop, and then injected onto the analytical column using a Rhodynne injector. Peptide samples were analyzed using a discontinuous gradient at a flow rate of approximately 10 μL / min generated by an Agilent 1100 series capillary pump. During recording, the flow was split into approximately 200 nL / min upstream of the injector. Analytical HPLC separation was performed using a C18 Zorbax 5 micron 300 angst filter. The procedure was performed using a ROHM (150 mm × 0.15 mm) frit-equipped capillary column. The acetonitrile profile was started at 5%, sloped to 12% after 5 minutes, then increased to 65% over approximately 90 minutes, remained at 65% for 5 minutes, decreased to 50% over 15 minutes, and then reduced to a final ratio of 5% before injection of the next step fraction from the same adult or fetal serum sample. NanoHPLC eluates were analyzed by detection by MS and fragmentation by MS / MS in a linear quadrupole ion trap (Schwartz, JC, MWSenko, and JESyka, A two-dimensional quadrupole ion trap Mass Spectrometer. J Am Soc Mass Spectrom, 2002.13(6):p.659~69). The device was configured to collect precursors for up to 200 milliseconds before MS / MS fragmentation in up to four MS / MS fragmentation cycles per precursor ion.

[0023] Correlation analysis

[0040] In this study, 1,554,347 precursor ions from fetal vs. adult MS / MS spectra were recorded by nanoLC-ESI-MS / MS. Correlation analysis of ion trap data was performed using X! TANDEM (Craig, R. and RCBeavis, TANDEM: matching proteins with tandem mass spectra. Bioinformatics, 2004.20(9):p.1466~7) and SEQUEST (Yates, JR, 3rd et al., Method to correlate tandem mass spectra of Tandem mass spectra were matched using the algorithm (Modified peptides to amino acid sequences in the protein database. Anal Chem, 1995. 67(8):p.1426-36) to peptide sequences from a library of 209,111 bovine proteins differing by at least one amino acid from RIKEN, IMAGE, RefSeq, NCBI, Swiss Prot, TrEMBLE, ENSEMBL, UNIPROT, and UNIPARC, along with available gene symbols, all previous accession numbers, description fields, and any other available annotations, made non-redundant by protein sequences in SQL Server. Identified peptides with any count of more than 10,000(E4) precursors were accepted into separate servers for each algorithm as complete trypsin-digested peptides and / or trypsin-digested phosphopeptides, and the results were combined and compared in SQL Server / R. A ±3 m / z X! TANDEM default ion trap data setting was used for precursor peptides considered to be from 300 m / z to 2000 m / z with a tolerance of 0.5 Da error in fragments. Peptides with the best fit of MS / MS spectra to complete trypsin digestion (TRYP) and / or trypsin digestion phosphopeptide (STYP) in +2 vs +3 charge states were accepted, including additional acetylation or oxidation of methionine and possible loss of water or ammonia.

[0024] Data analysis, transformation, and visualization

[0041] A linear quadrupole ion trap provided precursor ion intensity values ​​and peptide fragment MS / MS spectra correlated with specific trypsin-digested peptides (TRYP) or trypsin-digested phosphopeptides (STYP) using the X! TANDEM and SEQUEST algorithms. Protein accession numbers, actual and estimated peptide masses, correlated peptide sequences, peptide and protein scores, resulting protein sequences, and other relevant data were captured and assembled together in a SQL Server relational database. MS and MS / MS spectra, along with the results of the X! TANDEM and SEQUEST algorithms, were parsed into the SQL Server database, and overlapping fits of the MS / MS spectra were used for generic R data. Prior to graphical and statistical analysis in the system, the data was filtered to best fit. After correcting for the observed frequency of the number of MS / MS spectra recorded in the fetal vs. adult experiment, the peptide vs. protein correlation counts for each gene symbol were compared for fetal serum vs. adult serum using a chi-square test with Equation #1: i) (Fetus-Adult) 2 / (Adult+1) Formula #1 Precursor intensity data for MS / MS spectra are log 10 The data were converted, tested for normality, and analyzed by mean, standard error, and ANOVA across adult vs. fetal processing, fractions, and replicates (Bowden, P. et al., Quantitative statistical analysis of standard and human blood proteins from liquid chromatography, electrospray ionization, and tandem mass spectrometry. Journal of Proteome research, 2012.11: p. 2032 - 2047; Florentinus, A.K. et al., The Fc receptor-cytoskeleton complex from human neutrophils. Journal of proteomics, 2011.75: p. 450 - 468).

[0025] Cell culture

[0042] All culture media, blood samples, supplements, and reagents were sterilized before cell culture. Micropipettes, pipette tips, microcoverslips, and cell culture flasks / plates were autoclaved to create a sterile environment, and no antibiotics were used. 25 mL of FBS was added to 500 mL of DMEM (5% serum). 0.5 mL aliquots of 264.7 frozen third passages of RAW cells from ATTC were diluted in 3 mL of 5% FBS in DMEM in a cell culture flask and incubated to confluence in a humidified atmosphere of 5% CO2 at 37°C. Because the RAW cells contained DMSO when frozen in liquid nitrogen, the medium was changed after the first 4 hours. Cells were passaged and scraped when they reached 80% confluence. Effect of each serum on cell growth: Assays of three independent batches of FCS from different sources and three independent treatments of ABS from different sources were compared. Cells were seeded in 6-well plates containing up to 2 mL of 5% (v / v) FBS in DMEM and up to 2 mL of 5% (v / v) ABS in DMEM. Cells were cultured in an incubator at 37°C and sampled (passaged) every 48 hours. Cell growth rate was measured by observing cell confluence under a light microscope, and cell death and large cell breakdown in ABS were observed over time compared to cells in proliferating FCS. For hormone experiments, confluent 5th passaged cells were plated in medium containing 5% ABS at approximately 30%, and the amount of hormones shown was compared to that in 5% FCS. For cell size assays and imaging, the medium was removed, and each coverslip with attached cells was washed three times with 2 mL of 1×PBS. For cell fixation, 2 mL of 2% paraformaldehyde was added to each well for no more than 2 hours. Paraformaldehyde was removed, the cells were washed three times with 2 mL 1×PBS, then stained with rhodamine-phalloidin, and imaged under a Zeiss 520 metal laser confocal microscope.

[0026] result Cell morphology

[0043] Cells grown in fetal serum divided rapidly, spreading vertically upwards, as reflected by their small cell length, forming organized proliferative foci into spheres of cells stacked on top of each other. In contrast, cells cultured in adult serum formed elongated rhomboid monolayers with dendritic elongation that divided slowly and eventually died. Staining with rhodamine phalloidin revealed that fetal serum produced round, symmetrical cells with an average diameter of 10 microns, while adult serum produced elongated cells with a length of approximately 40 microns. This was shown to have occurred (Figure 1).

[0027] Stepwise fractionation of peptides and proteins

[0044] Proteins from fetal bovine serum compared to adult serum were separated on a ceramic quaternary amine resin, and the fractions were tested for protein content by Dumbroff dot blotting before degrading the proteins by tricine SDS-PAGE, which exhibits selectivity across salt gradients (Figures 2 and 3). Organic precipitates of endogenous peptides from fetal bovine serum compared to adult serum were separated by a water gradient and differential centrifugation, and the protein content was tested by the Dumbroff method before degrading the polypeptides on tricine SDS-PAGE, which exhibits selectivity for low molecular weight polypeptides (Ghosh, S. et al., Use of a scanning densitometer or an ELISA plate reader for measurement of nanogram amounts of protein in crude extracts from biological tissues. Anal Biochem, 1988. 169(2):p.227~33) (Figures 2 and 4). The optimal organic solvent composition for endogenous peptide extraction was 40% to 60% acetonitrile, while the optimal salt concentration for intact protein elution from quaternary amine resin was 100–175 mM NaCl (Table I).

[0028] [Table 1]

[0029] Normality and variability across processing and replication

[0045] Logs from all processes and replications. 10 The precursor intensity values, when combined, approached linear and Gaussian distributions (Figure 5A). The average results from six experiments (two treatments × three replicates) were comparable in terms of precursor peptide intensity obtained with and without considering phosphorylation (Figure 5B).

[0030] SQL Server Filtering

[0046] A pool of trypsin-digested peptides from proteins, as well as endogenous trypsin-digested (TRYP) peptides and / or trypsin-digested phosphopeptides (STYP), is obtained from fetal bovine serum (FBS) versus adult bovine serum (ABS) using liquid chromatography and nano- chromatography. Random and independent sampling of endogenous tryptic peptides from normal human peptides was performed without substitution by electrospray ionization and tandem mass spectrometry (LC-ESI-MS / MS) (Dufresne, J. et al., Random and independent sampling of endogenous tryptic peptides from normal human peptides). EDTA plasma by liquid chromatography, micro electrospray ionization, and tandem mass spectrometry. Clin Proteomics, 2017.14:p.41). Raw correlations from precursor ≥ E4 intensity counts were filtered in the SQL server to retain only the best fit by charge state and peptide sequence, completely avoiding the reuse of the same MS / MS spectrum. Serum LC-ESI-MS / MS recorded 1,553,347 MS / MS spectra, and X! TANDEM induced 61,152 trypsin digestion correlations (3.9%) (Table II).

[0031] [Table 2]

[0032] Peptide and protein identification

[0047] There was little agreement at the peptide level between peptidomics and proteomics, with 348,543 peptides observed only from exogenous digestion of proteins (proteomics) and 142,197 peptides observed only from endogenous peptides (peptidomics). Approximately 99.3% agreement was observed in proteins independently identified by peptidomics versus proteomics. 225,097 distinct endogenous peptide sequences from peptidomics were found to be 23% distinct from 431,443 distinct peptide sequences from proteomics. Although some duplication was observed, the set of identified proteins showed almost perfect agreement. Less than one-third of all bovine proteins were identified, along with at least five peptides. In contrast, the vast majority of the 183,426 known bovine protein accessions were not observed at all. This alone is sufficient evidence to demonstrate the truthfulness of LC-ESI-MS / MS of blood peptides using a simple ion trap (Bowden, P. et al., Meta sequence analysis of human blood peptides and their parent proteins. Journal of (Proteomics, 2010.73: pp. 1163-1175). However, although the two sets of observed peptide sequences were dramatically different, they originated from the same set in the parent protein: organic extraction of endogenous peptides (peptidomics) identified a set of 58,200 protein accessions that showed almost complete overlap and agreement with the set of 59,799 accessions from protein isolation and trypsin digestion (proteomics) (Table III).

[0033] [Table 3]

[0034] Proteins, accessions, and gene symbols of bovine serum

[0048] X! TANDEM identified approximately 12,000 protein gene symbols along with multiple peptides (Figure 7), while the SEQUEST algorithm provided observation frequency counts. After considering charge state and sequence fitting, the best-fitting MS / MS spectra of the peptides were subsequently analyzed by a generic R statistical system, showing that approximately 59,000 protein accessions and more than 20,000 protein gene symbols, along with at least 5 peptides, were detected by the sum of the X! TANDEM and SEQUEST results (Figure 6), which should have resulted in fairly certain identification. X! TANDEM enabled the calculation of cumulative p-values ​​and FDR-corrected q-values ​​for each gene symbol using the Benjamini and Hochberg method (Zhu PH, KK, Jankowski A, Marshall J, Comparison of published human serum / plasma data. Molecular and Cellular Proteomics, 2004.3(0.1):p.S235), and showed approximately 12,000 protein gene symbols with q-values ​​of 0.01 or less (Figure 8).

[0035] Fetal protein

[0049] A highly significant increase in the observed frequency in fetal serum (χ²) 2 Proteins showing a value of ≥50 are alpha-fetoproteins that are known to be expressed in the fetal liver and that show approximately 39% homology to BSA and 20% homology to vitamin D-binding protein according to BLAST. (Table IV) Common serum proteins specifically increased in fetal serum include alpha-2-HS glycoprotein precursor, serpin peptidase inhibitor clade A, fetuin B, gammaglobin, inter-alpha-trypsin inhibitor heavy chain H3, collagen and calcium-binding EGF domain-protein, pyruvate carboxylase, adenylate kinase, hemoglobin subunit beta, kallikrein K, thrombospondin 4, and ATP synthase membrane subunit (ATP5MD). Fetal serum was, as expected, rich in insulin-like growth factor II (Yao, T. and Y. Asayama, Animal-cell culture media:History,characteristics,and current issues.Reprod Med Biol, 2017.16(2):p.99~117).

[0036] [Table 4-1]

[0037] [Table 4-2]

[0038] AKT HRAS cell protein in plasma

[0050] Chi-squared analysis revealed several proteins with obviously too large χ² values ​​(χ²≧60, p<0.0001, df1) due to random sampling error (Figure 9). Chi-squared analysis also identified many obvious cellular proteins, including ligands, receptors, signaling proteins, kinases, phosphatases, G proteins, cyclases, G protein-related receptor enzymes such as phosphodiesterases, proteins with interaction domains such as the SH2 protein, as well as nucleic acid-binding proteins such as zinc fingers, histones, bromodomains, homeobox proteins, and transcription factors that can contribute to cell transformation (Marshall, J. et al., Creation of a federated database of blood proteins). ins: a powerful new tool for finding and characterizing biomarkers in serum.Clin Proteomics, 2014.11(1):p.3), all of them were clearly increased in fetal serum. X! showed a higher frequency, chi-squared (χ2) value of ≥9 in fetal serum. A complete list of proteins identified by TANDEM is shown in Table V. Examples of additional factors observed included nuclear receptor corepressor 2 isoform X2, atrial natriuretic peptide receptor 2, scavenger receptor class A member 3, ALK tyrosine kinase receptor, and many others.

[0039] [Table 5-1]

[0040] [Table 5-2]

[0041] Insulin vs. LEDGF

[0051] Based on proteins that increased in fetal bovine serum and showed significant χ2 values, and from the literature, the inventors selected insulin and LEDGF to be tested in a cell growth assay. (Previous results: Lieberman, I. and P. Ove, Growth factors for mammalian cells in culture. J) Consistent with Biol Chem (1959.234:p.2754~8), the addition of insulin to adult bovine serum restored some of the round, undifferentiated phenotype associated with rapid cell proliferation, but was not as effective as with fetal bovine serum. The IC50 of insulin was approximately 1 μg per mL (Figure 10). Similarly, the addition of lens epithelial cell-derived growth factor (LEDGF) alone or in combination with insulin partially restored the round phenotype, It was not as effective as complete fetal serum (Figure 11). Analysis revealed that fetal serum-specific growth factors, such as lens epithelial cell-derived growth factor (LEDGF), clearly control cell size and development. The results of the differential experiment were also consistent with the fidelity and sensitivity of LC-ESI-MS / MS of serum detecting small amounts of fetal serum-specific growth factors.

[0042]

[0052] This disclosure is described in conjunction with the specific embodiments shown above, but many of their substitutes, as well as their modifications and alterations, will be apparent to those skilled in the art. All such substitutes, modifications, and alterations are deemed to be within the scope of this disclosure. This specification encompasses the following inventions: [Item 1] A method for stimulating cell growth, comprising the step of bringing cells into contact with a composition containing isolated lens epithelial cell-derived growth factor (LEDGF). [Item 2] The method according to Item 1, wherein the composition is purified fetal serum. [Item 3] The method described in Item 1, further comprising hormones. [Item 4] The method according to Item 3, wherein the hormone is a growth-related hormone. [Item 5] The aforementioned hormones include insulin, insulin-like growth factor 2, multiple EGF-like domain 11 (MEGF11), erythropoietin (EPO), pigment epithelial cell-derived factor (PEDF), interleukin-17D (IL17D), interleukin-37 (IL37), interleukin-17C, interleukin-13, interleukin-2, retinol-binding protein 4 (RBP4), tissue factor pathway inhibitor (TFPI2), nerve growth factor beta polypeptide (NGFB), growth hormone somatotropin (GH1 / GH), multiple EGF-like domain 6 (MEGF6), tumor necrosis factor (TNF), and cardiotrophin-like hormones. The method according to item 3, selected from the group consisting of cytokine factor 1 (CLCF1), teratoma-derived growth factor (TDGF), glycoprotein hormone subunit beta 5 (GPHB5), cytokine receptor-like factor 1 (CRLF1), transforming growth factor beta (TGFB), growth hormone exon 5 (GHE5), CC motif chemokine ligand 21 (CCL21), platelet-derived growth factor (PDGF), platelet-derived growth factor receptor-like (PDGFRL), growth differentiation factor (GDF), fibroblast growth factor (FGF), CXC motif chemokine ligand (CXCL), and insulin-like growth factor-binding protein (IGFBP). [Item 6] The method according to any one of Items 1 to 5, wherein the cells are stem cells. [Item 7] The method described in any one of items 1-5, for the purpose of stimulating a greater number of cell division cycles. [Item 8] The method described in any one of items 1 to 5, for the purpose of extending the lifespan of the cells. [Item 9] The method according to any one of Items 1 to 5, wherein the step of bringing the cells into contact is performed during cell culture. [Item 10] The method according to any one of Items 1 to 5, wherein the step of bringing the cells into contact is in situ.

Claims

1. A method for stimulating cell growth, comprising the step of contacting cells with a composition containing isolated lens epithelial cell-derived growth factor (LEDGF).

2. The method according to claim 1, wherein the composition is purified fetal serum.

3. The method according to claim 1, further comprising a hormone.

4. The method according to claim 3, wherein the hormone is a growth-related hormone.

5. The aforementioned hormones include insulin, insulin-like growth factor 2, multiple EGF-like domain 11 (MEGF11), erythropoietin (EPO), pigment epithelial cell-derived factor (PEDF), interleukin-17D (IL17D), interleukin-37 (IL37), interleukin-17C, interleukin-13, interleukin-2, retinol-binding protein 4 (RBP4), tissue factor pathway inhibitor (TFPI2), nerve growth factor beta polypeptide (NGFB), growth hormone somatotropin (GH1 / GH), multiple EGF-like domain 6 (MEGF6), tumor necrosis factor (TNF), and cardiotrophin-like cytokines. The method according to claim 3, wherein a protein selected from the group consisting of chromosome factor 1 (CLCF1), teratoma-derived growth factor (TDGF), glycoprotein hormone subunit beta 5 (GPHB5), cytokine receptor-like factor 1 (CRLF1), transforming growth factor beta (TGFB), growth hormone exon 5 (GHE5), C-C motif chemokine ligand 21 (CCL21), platelet-derived growth factor (PDGF), platelet-derived growth factor receptor-like protein (PDGFRL), growth differentiation factor (GDF), fibroblast growth factor (FGF), C-X-C motif chemokine ligand (CXCL), and insulin-like growth factor-binding protein (IGFBP).

6. The method according to any one of claims 1 to 5, wherein the cells are stem cells.

7. The method according to any one of claims 1 to 5, for the purpose of stimulating a greater number of cell division cycles.

8. The method according to any one of claims 1 to 5, for the purpose of extending the lifespan of the aforementioned cells.

9. The method according to any one of claims 1 to 5, wherein the step of bringing the cells into contact is performed during cell culture.

10. The method according to any one of claims 1 to 5, wherein the step of bringing the cells into contact is performed in situ.

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