Patent CA-2610324-A1 (CA)
Inventor: VOON GERALD
IPC: C07K14/82
Filed: 2007-12-17
Abstract: We plan to combine (among others) use transcription factors Oct-4, Nanog and Sox2 to suppress genes to keep our stem cell inventory (proliferation strategy) and from differentiation and cell surface antigens including glycolipids SSEA3 and SSEA4 and the keratan sulfate antigens Tra-1-60 and Tra-1-81, all of the above to keep the stem cells in their stem cells forms (from differentiation) of pluripotency. We can also add telomerase.  We add to this process, oncogene c-Myc, especially if the cell colonies start to differentiate, the purpose is to reverse differentiate the cells into pluripotency state.   We then set up the medium we are considering several alternatives or combination (even all together).   1) Ectogenesis (eg. artificial uterus and fallopian tube cell) as medium for stems also. 2) Slowly and gently pumping in perflubron (oxygen supply) and nutrients, glucose (we could synthesize amino acids), such as those found in amniotic fluid. We need to come up with the best recipe of alanine, arginine, aspartic acid, isoleucine, cysteine, glutamic acid + glutamine, glycine, leucine, lysine, methionine, phenylalanine, proline, taurine, threonine, serine, valine (and possibly tyrosine) in and out in ebb and flow, including to flow to wash out waste.  Additionally we need to add NaHCO3 and possibly antibiotics (we could possibly emulsify egg yolk such as from a chicken; and also inorganic salts such as Na+, K+, Ca2+, Cu2+, Zn2+ and CO2+) as well as vitamins. 3) Other medium are made from (heat-inactivated possibly fetal; liver; spleen) Bovine (serum albumin), Lamb, pig, 2-mercaptoethanol...   4) Prepared Mediums include Dulbecco's (GIBCO; Eagle's), Krebs-Ringer bicarbonate medium as well as glucose, lactate, and pyruvate, Chocolate Agar, JRH-SFM 5) We also add Wnt (possibly micro inject into the nuclear membrane), Leukemia Inhibitory Factor (LIF), STAT 3, Fiberoblast Growth Factor (bFGF or FGF-2), Human Choronic Gonadotropin agonist leuprolide (GnRH) (peptide hormones), paracine - acting factor, Stem Cell Factor (SCF) building blocks (to establish receptors) for c-kit and mRNA increased successful functionality and also cytokine G-CSF and GM-CSF. We could also add Heparin-binding growth factors (eg. HBGF-1 and HGBF-2).   6) Other medium includes JRH Biosciences' Excel 620 serum-free medium (JRH-SFM). (Passaging of cells are used to further proliferate stem cells) Accutase (Innovative Cell Technologies Inc., distributed by PAA) and triturated approximately 10 times using a pipette.  Then, the cell suspension was incubated. Dissociated cells were centrifuged at 120 rcf and resuspended. An aliquot was counted by trypan blue exclusion assay in a hemocytometer to determine the amount of viable cells. Cells (10 5) were plated in T75 culture flasks for long-term passaging (culture medium (coated with fibronectin/lanmine) per flask). All media contained 1-glutamine, penicillin/streptomycin, heparin (Sigma), bFGF (R&D  Systems), and EGF (R&D Systems). A total of 10 4 cells per well were seeded in 12-well plates in a volume of 1 ml and grown under standard conditions. After a few days the grown stem cells were counted and dissociated by Accutase, and viable cells were counted by trypan blue exclusion assay in a hemocytometer.     In addition to substituting to male somatic DNA for female Oocyte nucleus we can see below...     Stem Cells from female eggs can be obtained as follows:  1) Treat the eggs with oncogene c-Myc. 2) Hyper ovulate (use of fertility drugs) 3) Harvest eggs. 4) Mature them in CMRL-1066 media (Sigma), FCS (HyClone), pregnant mare serum (Sigma), human chrionic gonadotropin (Sigma), penicillin, streptomycin. 5) The eggs are incubated in CO2, inonomycin, then in 6-dimethyllaminopurine. 6) The stem cells are then removed and further media are similar to the media above (stem cell tissue culture - especially proliferation; since before we can treat patients we need sizable inventory of compatible stem cells first). 7) We could use electroshock, heat shock to get the stem cells to start dividing. 8) We could also use the above maturation cycle and incubation to proliferate stem cells produced by the traditional method (eg. Oocyte removed nucleus, micro; laser;  electro shock and replaced with somatic DNA). 9) Alternatively we could use primordial germ cells from an adult male and use the above maturation cycle and incubation to proliferate stem cells. 10) Another alternative is to use a batch of stem cells (from any of the above methods) that have stopped proliferating and either treat with the maturation cycle, then incubate to jump start the proliferation cycle again. 11) Or we could use a batch of stem cells (from any of the above methods) and microinject, laser electroshock the DNA from the stem cells (that have stopped proliferating) into a fresh Oocyte (with the nucleus removed). 12) We could also alternate between 10) and 11) or test which one is the most prolific and which one is best for maintaining the integrity of the DNA - from mutating or degenerating.   We are looking at developing an fMRI or MRI (with adjustable pixelation to match the sharpness/detail as the magnification increases) - electron microscope (that can track the motion of tracker) and perhaps more important, as well the fMRI or MRI and/or PET also a tiny probe, lens similar to the video (real time) probes used by surgeons to pass through blood stream, except our video probe will be (eg, possibly with fiberoptic or diamond lens -due to the small size of the lens we need a lens that won't become brittle at that size) at the cellular level. It will enter the cell membrane via a prick of a needle and be able to probe around the inside the cell (we could also surround the probe with gel so the cell does not leak). The purpose for a video probe is to observe the interior of the cell in colour and carry out dye and stain phospherousent (fluorescence). We might include infrared capabilities in our probes. We use the dye and stain on inputs such as Oncogene c-Myc, different chemicals, different enzymes, different, growth factors, different amino acids, and also glucose and oxygen and carbon dioxide and differing levels wastes all in differing amounts (also salts) as well as all mediums, including maturing mediums, incubation mediums, passaging mediums, and ectogenesis mediums.     We observe which of these passes through the cell membrane:     1) By osmosis (hydrophobicity and lipophilicity process) what level of concentration of organic salts are optimal for cell health and their ability to carry other of the inputs above as they pass through the cell membrane. 2) Protein receptors which protein receptors bind to which inputs and how (mechanism) these receptors operate to bring the inputs in and deliver to the right user. 3) Why the protein receptors (bis-peptides) selects a particular input. 4) We can watch the cell organelles (internal structures - also DNA intake and outtake of chemicals entering and exiting the nuclear membrane and the ability of the nucleotides to sense feedback an how it releases which chemicals and which parts of the organelles react eg. mRNA to carry out instructions which nucleotides activate and deactivate consistently before a cell division and which deactivate when the cells stop proliferating) in terms of changing size, shape (observing possibly for real time pulsating or other indicators of activity) and location over various time horizons (what in the early eg. moment by moment, what in cytoplasm facilitates optimal proliferation versus late stage proliferation, why does the proliferation slow down even with growth factors, what and why is the cell make up eg. the organelles responding -reacting to to slowing down late stage proliferation) in real time (or video recorded) as an indication of activity of a particular organelle or of vertical chain requiring more than one type of organelle to put the input into a usable form - 1. are the output from the organelles' use of the input in a new form? 2. where is this output to be used? 3. How does it get there? 4. And what is its contribution to the process of the destined part of the cell and how is it used eg. signals that activate or deactivate a nucleotide, 5. What happens if you increase the concentration of the inputs outside the cell membrane, inside the cell membrane, inside the nuclear membrane. 5) We use phospherousent (fluorescence) colour stained/dyed to one or combination inputs to observe how they are delivered to which organelle, the rate of consumption by the amount delivered to the entry point(s) of an organelle and its change in concentration (as well the dye/stain being absorbed - appearing under the colour video probe) from higher to lower. We can note the output point(s) the form and amount per input guesstimate used/absorbed by biologically extrapolating and reverse engineering of the consumption/catalyst/usage (eg. communicate and instruct/give directions via DNA; telomerase changes why/how/how to prevent and how to repair; RNA such as maintenance, growth, proliferation, dormancy, differentiation). 6) What catalysts (enzymes) within the organelle process particular inputs and what outputs are produced. 7) Which outputs are used by other parts of the cells (how their delivery system operates). 8) Is there more than one vertical chain (An) of processes. 9) Is there a conduit of multiple processing (if there are layers of absorption and processing; what is their function). 10) How does the different organelles shape make for and Darwinian evolutionary survival advantage. 11) Are there multiple chambers to some of the organelles, is this for storage capacity or is it for a different processing function. 12) How does the cell know how to move which inputs and their processed products to the to further uptake processing to the place where they will be used/consumed. 13) We want to study the levels of levels and types processed outputs of prepared by the organelles and consumed for example when signalling a stem cell to continue proliferating or slow rate of proliferating (dormant cell activity).     14) The relative input absorption capacity/rate for example when signalling a stem cell to continue proliferating or slow rate of proliferating (dormant cell activity). 15) What role does gravity have to do with location (and size, shape - and proliferation rate) of organelles and nucleus (how any and all these factors affect function 0.5%) especially 3 - dimensional layout. Also the level concentration of fluid within. 16) We believe that cell division/proliferation is based on some condition in the cytoplasm, since somatic/adult DNA implanted into the Oocyte (with its own nucleus removed) in place of the Oocyte/egg's own nucleus provides the conditions inside (with a little help from electro shock) causes the somatic DNA Oocyte to divide. To study these mechanisms (we will also look at the part DNA, genes, nucleotides, play (reacting to feedback signals) in the proliferation process. As well we plan to study the function ribosome (eg. facilitation and transcription), vesicle, rough endoplasmic reticulum, golgi apparatus, cytoskeleton, smooth endoplasmic reticulum, mitochondria, vacuole, cytosol, lysosome, centriole. 17) We plan to use microscopic fMRI and MRI and PET and micro video probe to train in real time activation and deactivation timing of the genes which can work in unionson, eg. all needed genes (that cause proliferation to start, continue and also to avert stopping) activated or possibly cascading (one gene activated causes another gene to activate) or possibly that the relevant genes activate in a vertical chain, consequitively. Also there is the role of the gene and its interaction such as reacting or activated or facilitated by a fresh Oocyte's cytoplasm and/or the organelles within the cytoplasm. 18) We could also use the micro video probe and the micro fMRI and/or the MRI  to observe phospherousent (fluorescence), and observe the substance intake and released/expelled, into and out of the nuclear membrane and their effect on the DNA  (when the a specific substance has entered the nuclear membrane and the changes it causes on the DNA and the DNA's output's (eg. signals versus waste) effect on other parts of the cell - organelles, cytoplasm composition (eg.  proliferation). 19) And as further study of 18) and 19) we could isolate the DNA strings (with micro fMRI  and micro MRI and/or PET micro video probe), And take a video stream of substance inputs (eg. Wnt or growth factors or Oncogene c-Myc) as they enter, the nuclear membrane (how they are moved to a particular nucleuotide or the other way around) why they activate some nucleuotide and not others and why after some time (many proliferations), the same substance inputs do not cause the (possible scenario of cause) same genes to activate (eg. continue proliferation) or the cytoplasm (even isolate a specific organelle) no longer reacts to the genes the same way (proliferation stops). 20) We could even use the micro fMRI and MRI and video probe and PET to observe the reaction (caused by addition substance inputs) to and even dissect the DNA  strings (within the cell or within the nucleus membrane) at different points in time (short term) before, during stages and after the reaction to the substance inputs exposure to the DNA, by laying the DNA (or even possibly using facial recognition technology -to track nucleuotide of the coiled DNA strings within the cell and nuclear membrane) straight/flat, and recording which nucleuotide are change their characteristics (what is the natural (difference in characteristics) that a nucleuotide looks when activated and deactivated) what is the process, what types of catalyst (chemicals) are needed, are there differences in the way one nucleuotide from another catalyze (common versus different inputs) to activate or deactivate and are the catalyzation process from caused by one substance input to another different in and the reacting genes different in process despite yielding similar phenotypes (eg. augmenting proliferation).     21) Additionally by reverse engineering the genes that have been deactivated or activated using micro fMRI, MRI and PET and video probe (comparing genes at most active proliferation time) and seeing how input substances effect which of these genes, and how these genes direct/instruct the organelles and/or RNA, to keep instructing and reinitiating and carrying out the proliferation process and observing all the way over the long term until proliferation has stopped. By observing the change in characteristics of the specific relevant nucleuotides (and at most active proliferation versus how they differ at end of proliferation). 22) We need to understand what has changed since optimal proliferation until proliferation has stopped. Could it be genes reacting, instructed by chemicals (1) outside the cell being uptaken into the areas responsible for cell proliferation or stopping proliferation - the process of which is dicussed above or 2) in the cytoplasm), eg. to waste, lack of oxygen, starvation ... even telomerase and growth factors, don't seem to be a immortality viable, could we inject the Wnt directly into the nuclear membrane in case such factors are not being uptaken, or 3) could it be that the cytoplasmic composition or organelles degrade, lose responsiveness or naturally count age factor - (like an internal clock GP)(currently unknown to science) that affects the genes or 4) other genes (activated by other input substances) block the reproductive genes/nucleuotide. 23) We will use the micro fMRI and MRI and micro video probe and PET to track the cell's parts; DNA (in its natural environment and also removed dissected and while still fresh add such substances as Wnt to observe its reactions), organelles and cytoplasm (as well as sample the enzymes and chemicals... being produced and being added) to observation interactions and determine the functions of all these factors. We make these observations with a keen objective to tracking the difference in conditions of these factors during the 1. optimal proliferation of the cell and when the 2. stem cell stop proliferation, and the factor conditions of a 3. somatic cells and the factor conditions of 4. Oocyte. We are looking for observable conditions in physical characteristics. Since most cell functions are regulated by the DNA, we are looking for natural activation and/or deactivation and the way we do this so that we can track the timing of (action (eg. to chemical/enzymes... - reaction of more than one nucleuotide that activates at the same time, or one activating the other or the requirement of more than one to be activated simultaneously ; in real time) of cascading nucleuotide by using (special) micro video lens that tracks mass is trained on the straightened (laidout) DNA string of the 4. types of cells above, whose proliferation phenotypes we are sure of, but the mechanism of modus operandi are not known.