METHOD FOR STIMULATING THE GROWTH OF A CELL

FIELD OF THE DISCLOSURE

The present invention relates to a method for stimulating the growth of a cell; more particularly, to a method for stimulating the growth of a cell with lens epidermal dermal growth factor (LEDGF).

BACKGROUND OF THE DISCLOSURE

Fetal serum contains factors that promote changes in gene expression and permit the indefinite culture of cell lines such as RAW 264.7 murine 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 regulate cellular proliferation. However, the effect of growth hormones was inferior to that of fetal serum even after supplementation with albumin, transferin, amino acids or dilute 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 so all of the factors that permit indefinite cell growth have yet to be 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 expand clonal cell lines and may have therapeutic applications and have basic 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 has been significant efforts to create cell growth media that contains the types of growth factors contained in natural serum for use in cell culture experiments and attempts to enumerate the proteins of fetal serum from umbilical chords 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).

SUMMARY OF THE DISCLOSURE

Lens epidermal dermal growth factor is firstly identified from fetal serum and found to stimulate the growth of a cell.

The present disclosure provides a method for stimulating the growth of a cell comprising contacting the cell with a composition comprising an isolated lens epidermal dermal growth factor.

In one embodiment of the disclosure, the composition is a purified fetal serum.

In one embodiment of the disclosure, the composition further comprises a hormone.

In one embodiment of the disclosure, the hormone is a growth related hormone.

In one embodiment of the disclosure, the hormone is selected from the group consisting of insulin, insulin like growth factor 2, multiple EGF like domains 11 (MEGF11), erythropoietin (EPO), pigment epithelium-derived factor [PEDF, also known as serpin F1 (SERPINF1)], interleukin-17D (IL 1 7D), 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 domains 6 (MEGF6), tumor necrosis factor (TNF, preferably TNF alpha), cardiotrophin like cytokine factor 1 (CLCF1), teratocarcinoma-derived growth factor (TDGF, preferably teratocarcinoma-derived growth factor-1), glycoprotein hormone subunit beta 5 (GPHB5), cytokine receptor like factor 1 (CRLF1), transforming growth factor beta (TGFB, preferably transforming growth factor beta 2), growth hormone exon 5 (GHE5), C—C motif chemokine ligand 21 (CCL21), platelet derived growth factor (PDGF), platelet derived growth factor receptor like (PDGFRL), growth differentiation factor (GDF, preferably growth differentiation factor 15, 11, 6, 5 or 3), fibroblast growth factor (FGF, preferably fibroblast growth factor 18, 14 or 8), C—X—C motif chemokine ligand (CXCL, preferably C—X—C 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).

In one embodiment of the disclosure, the cell is a stem cell.

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

In one embodiment of the disclosure, the method is for prolonging the lifespan of the cell.

In one embodiment of the disclosure, the step of contacting the cell is in a cell culture. In one embodiment of the disclosure, the step of contacting the cell is in situ.

The present disclosure is described in detail in the following sections. Other characteristics, purposes and advantages of the present disclosure can be found in the detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A show the effect of fetal calf serum on the morphology of RAW 264.7 murine macrophages cultured in DMEM plus 5% fetal calf serum; FIG. 1B show the effect of adult serum on the morphology of RAW 264.7 murine macrophages cultured in DMEM plus 5% adult bovine serum; FIG. 1C shows the distribution of cell length in adult (ABS) versus fetal (FBS) serum. Ten cells were measured from 3 spots on 3 cover slips from 3 independent batches of fetal versus adult serum. The longest axis of the cell was measured. F-statistic: 307.1 on 3 and 156 DF, p-value: <2.2e-16.

FIG. 2 shows flow chart of the protein (proteomics) and endogenous peptides (peptidomics) isolation and analysis steps.

FIG. 3A shows the fractionation of bovine plasma by a step gradient of NaCl in 20 mM Tris pH 8.85 buffer in Dumbroff protein assay; FIG. 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.

FIG. 4A shows the sequential extraction of low molecular mass polypeptides from serum using a stepwise solubilization in organic/water (Dumbroff protein assay); FIG. 4B shows the sequential extraction of low molecular mass polypeptides from serum using a stepwise solubilization in organic/water. Lanes: 1, Molecular Weight Standard. Acetronitrile (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.

FIG. 5A shows the reproducibility of the fetal versus adult serum samples with the QQ plot showing the normality of the log 10 transformed intensity values; FIG. 5B shows the reproducibility of the fetal versus adult serum samples with the box plot showing the average log 10 intensity and variation in log 10 and 99% confidence interval for the adult versus fetal serum replicates. Treatments: 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.

FIG. 6A shows the total number of protein accessions from the Bovine protein library that were correlated by the X!TANDEM and SEQUEST algorithms combined (redundant protein accessions); FIG. 6B shows the total number of protein accessions from the Bovine protein library that were correlated by the X! TANDEM and SEQUEST algorithms combined (best correlation per gene symbol). The Bovine protein

FIG. 7A shows the protein accessions identified by the SEQUEST and X!TANDEM algorithms separately. The Bovine protein library contained 209,111 protein accessions.library contained 209,111 accessions. FIG. 7B shows the protein accessions identified by the SEQUEST and X! TANDEM algorithms separately. The Bovine protein library contained 209,111 protein accessions.library contained 209,111 accessions.

FIG. 8 shows the cumulative p-value and FDR corrected q-value of the non-redundant peptides per gene symbol computed from the X! TANDEM results using the R statistical system.

FIG. 9A shows the quantile plots of the corrected difference in observation frequency (Delta) and Chi Square values of the fetal versus adult serum. Tryptic peptide corrected difference (delta) in observation frequency; FIG. 9B shows the quantile plots of the corrected difference in observation frequency (Delta) and Chi Square values of the fetal versus adult serum. Tryptic peptide Chi Square x2; FIG. 9C shows the quantile plots of the corrected difference in observation frequency (Delta) and Chi Square values of the fetal versus adult serum. Tryptic and/or STYP corrected difference (delta) in observation frequency; FIG. 9D shows the quantile plots of the corrected difference in observation frequency (Delta) and Chi Square values of the fetal versus adult serum. Tryptic and/or STYP peptide Chi Square x2.

FIG. 10 shows the effect of insulin on the longest cell axis of RAW 264.7 macrophages in Adult Bovine Serum (ABS) versus Fetal Bovine Serum (FBS). The levels of insulin added each well of a six well culture dish are shown. The letters indicate statistically significant differences by the Tukey Kramer Honestly Significant Difference (HSD) Test

FIG. 11 shows the effect of insulin on the longest cell axis of RAW 264.7 macrophages in Adult Bovine Serum (ABS) versus Fetal Bovine Serum (FBS). The levels of insulin or Lens Epithelial Derived Growth Factor (LEDGF) added each well of a six well culture dish are shown. The letters indicate statistically significant differences by the Tukey Kramer Honestly Significant Difference (HSD) Test.

DETAILED DESCRIPTION OF THE DISCLOSURE

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meaning commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; however, in the event of any latent ambiguity, definitions provided herein take precedence over any dictionary or extrinsic definition.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

As used herein, the term “or” is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

The present disclosure provides a method for stimulating the growth of a cell comprising contacting the cell with a composition comprising an isolated lens epidermal dermal growth factor (LEDGF).

Fetal serum contains factors such as alpha fetoprotein (AFP), insulin, and insulin-like growth factor 2 that support the indefinite division and growth of cancerous cell lines. Though all of the peptides and proteins of fetal serum that regulate cell growth have yet to be elucidated. In some embodiments of the disclosure, the peptides of fetal, versus adult serum, were extracted in organic solvent and the proteins resolved on quaternary amine resin for tryptic digestions. The resulting peptides were analyzed by random and independent sampling with LC-ESI-MS/MS. Peptides and proteins were identified by the X!TANDEM and SEQUEST algorithms. Precursor and fragment intensity values with peptide and protein assignments were collected in SQL Server for analysis with the R statistical system. The 225,097 endogenous peptide sequences from peptidomics showed 23% overlap with the 431,443 distinct peptide sequences from proteomics. However the independent sets of peptides showed 99.3% agreement on a small set of protein gene symbols while most proteins were never observed. The agreement by independent methods was an unambiguous and clear demonstration that LC-ESI-MS/MS of blood plasma with a linear quadrupole ion trap shows low error rates of peptide and protein identification. Fetal serum showed increased observation frequency of peptides from proteins involved in the insulin and AKT HRAS pathways. Many growth factors including lens epithelium derived growth factor were specific to fetal serum. The addition of insulin and/or LEDGF to adult serum partially restored the rounded phenotype of rapidly dividing cells but was not as effective as fetal serum. All of the peptides and proteins in blood fluid may be enumerated in using a simple linear quadrupole ion trap with ≤1% error that revealed all the proteins specific to fetal serum that support the immortal growth of cells. Accordingly, one of the embodiment compositions is a purified fetal serum.

One ore more the hormones can be added to the composition of the present invention. In one embodiment of the disclosure, the hormone is a growth related hormone. Examples of the hormone include, but are not limited to, insulin, insulin like growth factor 2, multiple EGF like domain 11 (MEGF11), erythropoietin (EPO), pigment epithelium-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), cardiotrophin like cytokine factor 1 (CLCF1), teratocarcinoma-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 (PDGFRL), growth differentiation factor (GDF), fibroblast growth factor (FGF), C—X—C motif chemokine ligand (CXCL), and insulin like growth factor binding protein (IGFBP).

After in vitro or in vivo contacting the composition of the present invention with a cell, the growth of the cell can be stimulated. The cell growth stimulated by the composition of the present invention includes producing more cell division cycles or prolonging the lifespan of the cell. The in vitro contacting step is performed through a cell culture process.

The following examples are provided to aid those skilled in the art in practicing the present disclosure.

Examples
Materials and Methods
Materials

The HPLC was an Agilent 1100 (Santa Clara, CA, USA). The linear ion trap mass spectrometer was a LTQ XL (Thermo Electron Corporation, Waltham, MA, USA). Ceramic quaternary amine resin was from BioRad. C18 ZipTips were obtained from Millipore (Bedford, MA). C18 HPLC resin was from Agilent (Zorbax 300 SB-C18 5-micron, 300 Angstrom). Solvents were obtained from Caledon Laboratories (Georgetown, Ontario, Canada). Three independent batches of fetal calf 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, aliquot and re-frozen and thawed once before being used and the remainder discarded. LEDGF (Lens Epithelium Derived Growth Factor) recombinant protein was supplied from MyBioSource (San Diego, USA). LEDGF recombinant protein was produced from Mus musculus (mouse) cells in culture and 0.1 mg of LEDGF was re-suspended in sterile PBS at concentration of before use as described. Insulin, human recombinant was supplied from Sigma-Aldrich (Canada). Insulin, human recombinant was dissolved in sterile PBS to a concentration of before use. All other salts and reagents were obtained from Sigma-Aldrich-Fluka (St Louis, MO) except where indicated.

Protein Separation Over Quaternary Amine Resin

The proteins in 25 uL serum were diluted in 200 μL of 20 mM Tricine pH 8.8 binding buffer.

A new disposable preparative quaternary amine columns was created 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: p. 1143-1155). A 100 μL column of quaternary amine resin was struck in a 1.5 mL transfer pipette inside of 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): p. 36482). Quaternary amine resin in 50% slurry was packed by cutting off the bulb to act as a buffer reservoir and packing the tip with a 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: p. 1143-1155). The resin was equilibrated with binding buffer prior to introducing the sample by gravity. The resin was washed with 3 volumes of buffer prior to an increasing salt step-gradient. In order to avoid cross-contamination the preparative quaternary amine column was discarded after a single use. The samples were then digested with 1/100 wt/wt trypsin for 12 hours, reduced in 2 mM DTT for 30 minutes at 50° C. before digesting a second time with 1/100 wt/wt trypsin. The samples were quenched with 5% formic acid and dried.

Endogenous Peptide Extraction

Serum samples (200 μL) were precipitated with 9 volumes of acetonitrile (90% ACN) (Tucholska, M., et al., Endogenous peptides from biophysical and biochemical fractionation of serum analyzed by matrix-assisted laser desorption/ionization and electrospray ionization hybrid quadrupole time-of-flight. Analytical biochemistry, 2007. 370: p. 228-245) in 2 mL sample tubes followed by stepwise extraction of the pellet. The acetonitrile/water extract was separated from the insoluble pellet at each step fraction with a centrifuge at 12,000 RCF for 5 minutes. The organic precipitate (pellet) that contains a much larger total amount of endogenous polypeptides was manually re-suspending in steps of increasing water content to yield 10 fractions from 90% ACN to 10% ACN, followed by water and then 5% formic acid (Dufresne, J., et al., A method for the extraction of the endogenous tryptic peptides (peptidome) from human EDTA plasma. Anal Biochem, 2018). The acetonitrile/water phase that contains peptides was collected, transferred to a fresh sample tube, dried under vacuum in a rotary lyophilizer, and stored at −80° C. for subsequent analysis.

Preparative C18 Chromatography of Peptides

Preparative C18 separation provided the best results for peptide and phosphopeptide analysis in a “blind” comparison (Krokhin, O. V., W. Ens, and K. G. Standing, 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). A new disposable C18 preparative “Zip Tip” column was used to collect each sub-fraction. Solid phase extraction with C18 for LC-ESI-MS/MS was performed as previously described. The C18 chromatography resin (Zip Tip) was wet with 65% acetonitrile before equilibration in water with 5% formic acid. The plasma extract was dissolved in 200 μL of 5% formic acid in water. The resin was washed with at least five volumes of the same binding buffer. The resin was eluted with >3 column volumes of 65% acetonitrile (2 μL) in 5% formic acid. In order to avoid cross-contamination the preparative C18 resin was discarded after a single use.

Analytical LC-ESI-MS/MS

In order to entirely prevent any possibility of cross contamination between serum samples, a new disposable nano analytical HPLC column and nano emitter were fabricated for recording each set of quaternary amine (proteomics) and organic (peptidomic) extractions (n=3 fetal plus n=3 adult 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=26 fractions per sample) for a total of 156 LC-ESI-MS/MS experiments (2 treatments×3 replicates×26 fractions). The ion traps were cleaned and tested for sensitivity with angiotensin and glu-fibrinogen prior to recordings (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: p. 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: p. 113680). Each disposable C18 analytical column was conditioned and quality controlled with a mixture of three non-human protein standards using a digest of Bovine Cytochrome C, Yeast alcohol dehydrogenase (ADH) and rabbit Glycogen Phosphorylase B to confirm the sensitivity and mass accuracy of the system (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). The conditioned column was extensively washed in 50% acetonitrile before introducing the sample fraction set. The stepwise fractions were collected over C18 preparative micro columns, eluted in 24, of 65% ACN and 5% formic acid, diluted with 18 μL of 5% formic acid in water and immediately loaded manually into a 20 μL metal sample loop before injecting onto the analytical column via a Rhodynne injector. The peptide samples were analyzed over a discontinuous gradient at a flow rate of −10 uL per minute generated with an Agilent 1100 series capillary pump and split upstream of the injector during recording to about −200 nL per minute. The analytical HPLC separation was performed with a C18 Zorbax 5 micron 300 Angstrom (150 mm×0.15 mm) flitted capillary column. The acetonitrile profile was started at 5%, ramped to 12% after 5 minutes and then increased to 65% over −90 minutes, remained at 65% for 5 minutes, decreased to 50% for 15 minutes and then declined to a final proportion of 5% prior to injection of the next step fraction from the same adult or fetal serum sample. The nano HPLC effluent was analyzed by ESI ionization with detection by MS and fragmentation by MS/MS with a linear quadrupole ion trap (Schwartz, J. C., M. W. Senko, and J. E. Syka, A two-dimensional quadrupole ion trap mass spectrometer. J Am Soc Mass Spectrom, 2002. 13(6): p. 659-69). The device was set to collect the precursor for up to 200 milli seconds prior to MS/MS fragmentation with up to four MS/MS fragmentations per precursor ion.

Correlation Analysis

In this study 1,554,347 precursor ions from fetal versus adult MS/MS spectra were recorded by nano LC-ESI-MS/MS. Correlation analysis of ion trap data was performed with X!TANDEM (Craig, R. and R. C. Beavis, TANDEM.•matching proteins with tandem mass spectra. Bioinformatics, 2004. 20(9): p. 1466-7) and SEQUEST (Yates, J. R., 3rd, et al., Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database. Anal Chem, 1995. 67(8): p. 1426-36) algorithms to match tandem mass spectra to peptide sequences from a library of 209,111 bovine proteins that differed 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 annotation that was rendered non-redundant by protein sequence in SQL Server. Identified peptides with precursors greater than 10,000 (E4) arbitrary counts were accepted as fully tryptic peptides and/or tryptic phosphopeptides on separate servers for each algorithm and the results combined, and compared in SQL Server/R. The X!TANDEM default ion trap data settings of ±3 m/z from precursors peptides considered from 300 to 2000 m/z with a tolerance of 0.5 Da error in the fragments were used. The best fit peptide of the MS/MS spectra to fully tryptic (TRYP) and/or tryptic phosphopeptides (STYP) at charge states of +2 versus +3 were accepted with additional acetylation, or oxidation of methionine and with possible loss of water or ammonia.

Data Analysis Transformation and Visualization

The linear quadrupole ion trap provided the precursor ion intensity values and the peptide fragment MS/MS spectra that were correlated to specific tryptic peptides (TRYP) or tryptic phosphopeptides (STYP) by the X! TANDEM and SEQUEST algorithms. The protein accession numbers, actual and estimated peptide masses, correlated peptide sequences, peptide and protein scores, resulting protein sequences and other associated data were captured and assembled together in an SQL Server relational database. The MS and MS/MS spectra together with the results of the X! TANDEM and SEQUEST algorithms were parsed into an SQL Server database and redundant fits of MS/MS spectra were filtered to the best hit before graphical and statistical analysis with the generic R data system. After correcting the observation frequency for the number of MS/MS spectra recorded in fetal versus adult experiments, the peptide to protein correlation counts for each gene symbol were compared for fetal versus adult serum using the Chi Square test using equation #1:

i) (Fetal−Adult){circumflex over ( )}2/(Adult+1) EQN #1

The precursor intensity data for MS/MS spectra were logio transformed, tested for normality and analyzed across the adult versus fetal treatments, fractions, and replicates by means, standard errors and ANOVA (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).

Cell Culture

All media, blood samples, supplement and reagents were sterilized prior to cell culture. Micropipettors, pipette tips, micro cover glasses, and cell culture flasks/plates were autoclaved in order to create an aseptic environment and no antibiotic were employed. A volume of 25 mL FBS was added to a 500 mL DMEM (5% serum). A 0.5 mL aliquot of frozen 3^rdpassage of raw cells 264.7 from ATTC were diluted with 3 mL 5% FBS in DMEM in a cell culture flask and incubated in a humidified atmosphere of 5% CO2 at 37° C. until confluent. Media was changed after the first four hours since the raw cells contained DMSO when it was frozen down in the liquid nitrogen. Cells were passaged and scraped when they became 80% confluent. The effects of each serum on cell growth: three independent batches of FCS from different sources and three independent treatments of ABS from different sources assay were compared. Cells were seeded in 6-well plates with maximum 2 mL of 5% (v/v) FBS and 5% (v/v) ABS in DMEM respectively. The cells were cultured in the incubator at 37° C. and sampled (passaged) every 48 hours. The rate of cell growth was measured by looking at the confluence of the cells under a light microscope and the cell death and disintegration of large cells in ABS over time noted compare to cells in FCS that proliferated. For hormone experiments, confluent 5^thpassage cells were plated at −30% in medium with 5% ABS and the amount of hormone indicated compared to 5% FCS. For cell size assays and imaging the media was removed and each cover slip with cells adhered to it was washed 3 times with 2 mL 1×PBS. 2 mL of 2% Para-formaldehyde was added to each well for cell fixation for no more than 2 hours. Para-formaldehyde was removed and the cells were washed 3 times with 2 mL 1×PBS before staining with Rhodamine phalloidin and imaging under a Zeiss 520 Metal laser confocal microscope.

Results
Cell Morphology

The cells grown in fetal serum divided rapidly, as reflected by small cell length, and formed foci organized into a globe of cells that extended vertically upwards piled onto each other. In contrast, cells cultured in adult serum formed a monolayer of elongated rhomboids with dendritic extensions that divided slowly and eventually died. Staining with rhodamine phalliodin showed that fetal serum resulted in rounded symmetrical cells that average 10 microns across while adult serum produced elongated cells that were about 40 microns in length (FIG. 1).

Stepwise Fractionation of Peptides and Proteins

The proteins of fetal calf serum compared to adult serum were separated over ceramic quaternary amine resin and the fractions tested for protein content by the Dumbroff dot blotting method prior to resolving the proteins by tricine SDS-PAGE that showed selectivity over the course of the salt step gradient (FIGS. 2&3). The organic precipitate of endogenous peptides from fetal calf serum compared to adult serum were separated by a water step gradient and differential centrifugation, tested for protein content by the Dumbroff method (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) before resolving the polypeptides on tricine SDS-PAGE that showed selectivity for low molecular mass polypeptides (FIGS. 2&4). The optimal organic solvent composition for endogenous peptide extraction was from 40 to 60% acetonitrile while the optimal salt concentration for intact protein elution from quaternary amine resin was 100 to 175 mM NaCl (Table I).

TABLE I

Protein separation over the salt fractions versus endogenous

peptide separation over organic water on the proteins

and tryptic peptides identified (See FIG. 2).

Peptides

Fraction
Concentration
Redundant
Distinct
Distinct

Number
(%)
Protein
Protein
Peptide

1
0% AcN
223397
44397
48853

2
5% AcN
249899
46250
55977

3
10% AcN
250107
45728
53953

4
20% AcN
236723
44892
49899

5
30% AcN
249846
46750
58077

6
40% AcN
263797
48017
63652

7
50% AcN
271287
47568
62042

8
60% AcN
266640
47248
62155

9
70% AcN
278624
46975
59369

10
90% AcN
274823
46150
54845

463975
568822

Proteins

Fraction
Concentration
Redundant
Distinct
Distinct

Number
(mM)
Protein
Protein
Peptide

1
0
430748
51459
87107

2
50
393872
50332
79094

3
100
517203
52308
91084

4
150
478902
52205
93276

5
175
530335
53440
109606

6
200
379348
50820
83834

7
225
324976
50150
78085

8
250
278455
48049
64995

9
300
382052
50746
81509

10
350
351982
49386
73052

11
400
326096
48878
69684

12
450
261822
46261
57871

13
500
404293
50257
79141

14
600
322479
48271
67073

15
BdDigest
211181
43258
46105

16
QF
399748
50099
77835

795919
1239351

Normality and Variation Across Treatments and Replicates

The logio precursor intensity values from all treatments and replicates together approached a linear and Gaussian distribution (FIG. 5A). The average results of the 6 experiments (2 treatments×3 replicates) were comparable in terms of the intensity of the precursor peptides obtained with and without the consideration of phosphorylation (FIG. 5B).

SQL Server Filtering

The pool of tryptic peptides from proteins and endogenous tryptic (TRYP) peptides and/or tryptic phosphopeptides (STYP) were randomly and independently sampled without replacement by liquid chromatography, nano 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 EDTA plasma by liquid chromatography micro electrospray ionization and tandem mass spectrometry. Clin Proteomics, 2017. 14: p. 41) from fetal bovine serum (FBS) versus adult bovine serum (ABS). The raw correlations from precursors >E4 intensity counts were filtered to retain only the best fit by charge state and peptide sequence in SQL Server to entirely avoid re-use of the same MS/MS spectra. The LC-ESI-MS/MS of serum recorded 1,553,347 MS/MS spectra resulting in 61,152 tryptic correlations (3.9%) by the X!TANDEM (Table II).

TABLE II

The filtering of the MS/MS spectra and the resulting correlations to peptides to ensure that

only the best hit was accepted. The total number of MS/MS spectra of greater than E4 counts

collected in this study was 526,870 MS/MS spectra from organic extraction and 1,027,477 from

quaternary amine fractionation of proteins followed by digestion resulting in a total of

1,554,347 MS/MS spectra. Treatments: TRYP, fully tryptic with <3 missed cleavages; TRYP

STYP, fully tryptic with optional phosphorylation of serine, threonine or tyrosine. The Bovine

protein library contained 209,111 proteins that differed by at least 1 amino acid.

Total
Redundant
Distinct

Distinct
Distinct

Redundant
Spectra
MSMS
MSMS
Protein
Protein
Peptide

MS/MS
Count
Spectra
Spectra
Identifications
Identifications
Identifications

TRYP
2616468
36560334
2353004
36560334
204332
2777338

TRYP
2616468
36297856
2351922
36297856
201220
2598348

STYP

total
Redundant
Distinct

Distinct
Distinct

Best Fit
spectra
MSMS
MSMS
Protein
Protein
Peptide

Spectra
count
Spectra
Spectra
Identifications
Identification
Identifications

TRYP
2616468
7782701
2353004
7782701
179653
1063402

TRYP
2616468
7730807
2351922
7730807
176176
1040077

STYP

TOTAL
5232936

Redundant
Distinct

Distinct
Distinct

Best Fit

MSMS
MSMS
Protein
Protein
Peptide

Algorithm
Peptide
Spectra
Spectra
Identifications
Identifications
Identifications

X!TANDEM
TRYP
383535
127250
383535
57308
61152

X!TANDEM
TRYP
342083
116827
342083
44146
57435

STYP

SEQUE ST
TRYP
7399166
2344666
7399166
178895
1021440

SEQUE ST
TRYP
7388724
2345002
7388724
176133
998966

STYP

Peptide and Protein Identification

There was little agreement at the level of peptides from peptidomics versus proteomics with 348,543 peptides observed only from exogenous digestion of proteins (proteomics) and 142,197 peptides only observed from endogenous peptides (peptidomics). There was −99.3% agreement on the proteins independently identified by peptidomics versus proteomics. The 225,097 distinct endogenous peptide sequences from peptidomics showed 23% overlap with the 431,443 distinct peptide sequences from proteomics but showed near perfect agreement on the set of proteins that were identified. Less than one third of all bovine proteins were identified with at least 5 or more peptides. In contrast, the majority of the known 183,426 bovine protein accessions were never observed. That alone is sufficient evidence to demonstrate the veracity of LC-ESI-MS/MS of blood peptides with a simple ion trap (Bowden, P., et al., Meta sequence analysis of human blood peptides and their parent proteins. Journal of proteomics, 2010. 73: p. 1163-1175). However even though the two sets of peptide sequences observed differed dramatically, they were derived from the same set of parent proteins: organic extraction of endogenous peptides (peptidomics) identified a set of 58,200 protein accessions that showed near perfect overlap and agreement with the set of 59,799 accessions from protein separation and tryptic digestion (proteomics) (Table

TABLE III

Comparison of organic extraction of peptides versus separation

of proteins over quaternary amine chromatography. The redundant

and distinct proteins and peptides and the overlap between

treatments were computed in SQL Server (see FIG. 2).

Total
Organic
Salt
Common
Found
Found

Distinct
Distinct
Distinct
Distinct
Only in
Only in

Proteins
Proteins
Proteins
Proteins
Organic
Salt

60233
58200
59799
57766
434
2033

Total
Organic
Salt
Common
Found
Found

Distinct
Distinct
Distinct
Distinct
only in
Only in

peptides
peptides
peptides
Peptides
Organic
Salt

573640
225097
431443
82900
142197
348543

The Protein, Accessions and Gene Symbols of Bovine Serum

X!TANDEM identified some 12,000 proteins gene symbols with multiple peptides (FIG. 7) while the SEQUEST algorithm provided the observation frequency count. The peptide that was the best fit of MS/MS spectra after considering the fit of charge state and sequence were then analyzed by the generic R statistical system showed that −59,000 protein accessions and more than 20,000 protein gene symbols were detected by the sum of the X! TANDEM and SEQUEST results with at least 5 peptides (FIG. 6) that should confer near certain identification. X!TANDEM permitted computation of cumulative p-values and FDR corrected q-values for each gene symbol by the method of Benjamini and Hochberg (Zhu P-H, K. K., Jankowski A, Marshall J, Comparison of published human serum/plasma data. Molecular and Cellular Proteomics, 2004. 3(0.1): p. 5235) that showed some 12,000 protein gene symbols with a q-value of 0.01 or less (FIG. 8).

Fetal Proteins

Proteins that showed highly significant increases in observation frequency in the fetal serum (x2 value >50) included alpha fetoprotein that is known to be expressed in the fetal liver and shows about 39% homology with BSA and 20% homology to vitamin D binding protein by BLAST (Table IV). Common serum proteins specifically increased in fetal serum include alpha-2-HS-glycoprotein precursor, serpin peptidase inhibitor Glade A, fetuin B, gamma globin, 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). The fetal serum was enriched in Insulin-like Growth Factor II as expected (Yao, T. and Y. Asayama, Animal-cell culture media: History, characteristics, and current issues. Reprod Med Biol, 2017. 16(2): p. 99-117).

TABLE IV

Fetal serum specific proteins detected by fully tryptic peptides and fully tryptic

serine, threonine or tyrosine phosphopeptides from X!TANDEM and/or SEQUEST that

show a corrected observation frequency difference (Delta) and Chi Square (x2) value

of >50 for both tryptic (TRYP) and phosphotyptic (STYP) peptides.

Gene_Symbol
Count
TANDEM
SEQUEST
TRYP_X2
TRYP_Delta
STYP_X2
STYP_Delta

AHSG
13062
7364
5698
82229
6367
35346
2769

SERPINA1
6313
3602
2711
39176
3008
21045
1429

FETUB
1256
575
681
29189
704
5503
235

ITIH3
1663
757
907
8339
729
1733
303

AFP
2472
1222
1250
3512
903
948
351

MEPCE
4081
6
4075
2641
1352
14
22

LOC 104974567
6646
4
6642
2475
1125
2295
1098

PDCD4
6731
2
6729
2459
1125
2230
1104

DGAT1
10513
6
10507
1894
1292
2494
1749

RRAGB
432
6
426
1776
146
1921
131

SH2B1
5892
11
5881
1763
866
2185
1121

SPRY4
2836
14
2822
1626
551
1421
604

KIRREL3
5220
0
5220
1397
739
1919
975

FBLL1
3615
125
3490
1187
546
862
505

CCDC9
6014
20
5994
1180
683
2005
1153

NFATC2IP
5805
18
5787
977
594
1654
1077

SFPQ
5838
31
5807
906
568
1643
1089

HMX1
5729
19
5710
904
566
1634
1070

L2HGDH
2991
0
2991
899
445
825
1499

GNPTG
575
16
559
865
140
405
136

BRWD3
6084
10
6074
846
575
1583
1081

FGA
461
160
301
778
146
141
57

PLA2G2A
4547
4
4543
768
556
1160
797

LOC 101902853
262
0
262
762
78
367
74

LOC 100125947
4549
4
4545
762
555
1165
798

ADGRV1
4428
26
4402
757
512
1351
830

LOC788663
162
2
160
720
71
26
14

CTR9
6140
27
6113
687
546
1472
1056

MORF4L1
3149
2
3147
637
434
613
458

NXNL2
2112
4
2108
606
313
591
354

H2AFX
2581
6
2575
603
333
718
434

LOC100138633
596
8
588
598
129
338
134

RBMX
3505
28
3478
578
351
947
632

ETFA
1652
2
1650
558
190
1323
442

GAR1
3450
24
3426
478
313
818
596

ZRSR2
1225
8
1217
475
317
20
28

PPP1R12B
2121
10
2111
469
247
763
440

EME2
1947
4
1943
431
247
616
383

NFKBIL1
1599
13
1586
419
231
408
264

SUSD4
122
0
122
384
36
205
25

LUM
396
168
228
369
121
127
56

BDKRB1
1477
2
1475
368
183
446
270

IL2RA
337
1
336
350
110
25
17

LOC101902766
441
0
441
349
120
440
81

AKT1
2310
8
2302
347
269
365
307

MEETTL25
2270
4
2266
345
267
300
286

PRDM11
463
216
247
335
89
115
67

BAG4
235
0
235
329
44
204
59

FBLN1
554
86
468
319
112
276
105

FABP1
56
12
44
318
18
43
11

LOC787241
235
0
235
316
56
211
58

ITIH2
4294
2200
2095
312
537
74
188

CCDC124
1475
0
1475
304
185
465
264

MCAT
375
203
172
293
80
126
71

IK
586
218
368
290
138
45
46

FLJ10922
604
4
600
290
95
257
120

KIAA2012
1116
14
1102
287
158
53
78

TMEM143
611
4
607
286
95
241
119

KEAP1
1264
3
1261
274
158
491
238

TTLL10
1124
5
1119
270
155
243
168

MR VI1
1436
9
1426
263
164
477
246

ACOT6
223
123
100
257
54
113
43

LEO1
3222
2
3220
256
206
308
409

TMEM114
827
8
819
255
84
1007
275

LGMN
439
8
431
246
115
45
31

AHDC1
5051
43
5008
240
214
1662
1149

AMBP
1045
403
642
238
233
41
52

FUT2
218
0
218
237
45
312
53

AKT HRAS Cellular Proteins in Plasma

The Chi Square analysis showed some proteins with x2 values that were apparently far too large (x2≥60, p<0.0001, d.f. 1) to be resulted from random sampling error (FIG. 9). Chi Square analysis also identified many apparent cellular proteins such as ligands, receptors, signaling proteins, g-protein related, receptor enzymes like kinases, phosphatases, g-proteins, cyclases, phosphodiesterases, and proteins with interaction domains like SH2 protein as well as nucleic acid binding proteins including zinc fingers, histones, bromodomains, homeobox proteins and transcription factors that might contribute to cellular transformation (Marshall, J., et al., Creation of a federated database of blood proteins: a powerful new tool for finding and characterizing biomarkers in serum. Clin Proteomics, 2014. 11(1): p. 3), that were all apparently increased in fetal serum. The full list of proteins that were identified by X! TANDEM that showed greater frequency in fetal serum Chi Square (x2) values of >9 are shown in Table V. Additional example factors that were 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.

TABLE V

The growth factors, cytokines, chemokines, interleukins and tumor necrosis factor that differ between

fetal versus adult serum by Chi Square analysis of the tryptic (TRYP) and or serine, threonine

or tyrosine phosphorylated tryptic peptides (STYP) identified by SEQUEST or X!TANDEM where

X2 of fully tryptic (TRYP) or phospho tryptic (STYP) peptides are greater than 4 (p < 0.05).

Gene_Symbol
Count
X!TANDEM
SEQUEST
TRYP_Delta
TRYP_X2
Delta_STYP
X2_STYP

CCL20
11
0
11
2
5
0
0

CCL21
47
2
45
5
6
0
0

CGRRF1
28
0
28
6
17
0
0

CRLF1
146
37
109
11
10
9
4

CXCR1
103
0
103
15
41
2
1

CXCR2
51
0
51
6
11
1
0

DOCK11
241
7
234
9
5
−4
1

EGR2
44
0
44
7
9
−3
1

EGR4
148
4
144
2
1
29
34

FGF14
73
0
73
−9
6
−15
9

FGF4
33
0
33
−5
2
1
5

FGF7
60
1
59
−8
4
−1
0

Fgf8
60
0
60
−12
8
−3
1

GDF11
690
6
684
−48
18
−47
15

GDF11
690
6
684
−48
18
−47
15

GDF5
318
8
310
−9
2
−47
24

GDF6
140
2
138
7
4
13
9

GPRIN1
668
54
614
44
20
2
0

GPRIN2
54
4
50
−2
1
5
6

IGF2
172
6
166
38
57
8
5

IGFBP2
45
0
45
2
4
6
17

IGFBP4
199
7
192
26
27
−4
1

ILlORB
80
1
79
13
18
6
9

IL17C
87
0
87
−5
2
14
12

IL17RC
98
1
97
6
7
4
4

IL18RAP
158
6
152
13
15
−2
0

ILIA
28
2
26
0
0
3
4

IL1B
82
2
80
−2
0
−8
5

IL22RA2
59
2
57
7
6
−1
0

IL2RA
113
0
113
36
114
6
8

IL2RA
113
0
113
36
114
6
8

IL36A
23
2
21
3
4
−1
0

L6
49
0
49
−5
3
8
8

IRAK1BP1
28
1
27
2
2
3
5

Irak4
269
2
267
22
19
16
6

IRAK4
130
1
129
8
8
6
2

Irak4
269
2
267
22
19
16
6

LOC 100848143
68
0
68
1
0
10
9

LOC510185
58
2
56
10
19
1
0

LOC784541
128
1
127
33
77
4
2

LTBP4
184
4
180
8
7
−2
0

MEGF11
108
1
107
12
7
1
0

NFIL3
134
0
134
10
8
−1
0

OGFR
383
4
379
−22
4
−15
5

OGFR
383
4
379
−22
4
−15
5

OSGIN2
93
1
92
6
5
10
8

PDGFC
76
5
71
5
4
1
0

PDGFD
59
0
59
6
5
2
1

PDGFRL
109
2
107
−6
2
12
13

PRP6
51
4
47
5
4
−5
3

RERG
59
1
59
−2
1
−13
8

SOCS5
133
2
131
−11
5
8
16

TGFB2
113
0
113
12
16
−7
3

TMEM219
16
0
16
−1
0
3
26

TNF
45
3
42
8
9
2
1

TNFa
59
5
54
12
11
2
1

TNFAIP8L1
45
2
43
5
12
−2
0

TNFAIP8L2
79
2
77
11
29
13
20

TNF-alpha
24
5
19
1
0
4
7

TNFR2
9
0
9
4
16
0
0

TNFRSF13 B
91
1
90
−6
2
−19
13

TNFRSF1B
83
0
83
−9
6
−20
13

TNFRSF25
99
2
98
18
34
0
0

TNFSF12
52
2
49
6
5
1
2

TNFSF18
27
0
27
1
1
−5
4

TNFSF4
23
0
23
4
4
−2
1

TRAF1
141
0
141
−3
1
24
54

USMGS
242
0
242
65
108
4
15

Insulin vs LEDGF

From the proteins that were increased in fetal bovine serum and showed significant x2 values and the literature, we selected insulin and LEDGF to test in a cell growth assay. In agreement with previous results (Lieberman, I. and P. Ove, Growth factors for mammalian cells in culture. J Biol Chem, 1959. 234: p. 2754-8), the addition of insulin to the adult bovine serum restored some of the rounded, non-differentiated phenotype associated with rapid cellular proliferation but was not as effective as fetal bovine serum. The IC50 of insulin was approximately 1 μg per mL (FIG. 10). Similarly, the addition of Lens Epitheliam-Derived Growth Factor (LEDGF) alone, or together with insulin, partially restored the rounded phenotype but was not as effective as complete fetal serum (FIG. 11). The analysis revealed growth factors specific to fetal serum such as Lens Epidermal Derived Growth Factor (LEDGF) apparently regulates cells size and development. The results of the add-back experiment were also consistent with the fidelity and sensitivity of LC-ESI-MS/MS of serum to detect low abundance growth factors specific to fetal serum.

While the present disclosure has been described in conjunction with the specific embodiments set forth above, many alternatives thereto and modifications and variations thereof will be apparent to those of ordinary skill in the art. All such alternatives, modifications and variations are regarded as falling within the scope of the present disclosure.

	Number	Date	Country
Parent	PCT/CN2021/110473	Aug 2021	US
Child	18162807		US

METHOD FOR STIMULATING THE GROWTH OF A CELL

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

International Classifications

Abstract

Description

Claims

Provisional Applications (1)

Continuations (1)