Patent CA-2613026-A1 (CA)
Title: Added new stem cells processes/methods/techniques
Inventor: VOON GERARD
IPC: A61K35/12
Filed: 2008-01-15
Abstract: We want to use the product Dermatix or some equivalent product to 1) try to proliferate stem cells and 2) use it for scarred tissue inside the body a procedure done before grafting or adding or scaffolding stem cells (eg. into a spinal chord injury patient) so the stem cells aren't blocked from taking to injured wound.     Taken from Dermatix website:  The Innovation Dermatix.TM. Gel is a patented proprietary formula containing bio-inert and biocompatible silicone compounds, namely polysiloxane, silicon dioxide and nonvolatile silicone components. It uses basic long-chain polymers as topical silicone-gel sheeting. The result is a waterproof, gas-permeable membrane that acts like an extra layer of skin. It helps to soften and flatten the scar and to reduce pain, itching and scar-associated discoloration, while maintaining the moisture balance and elasticity of the adjacent skin. We are looking at running electrode through stem cell colonies to deliver electrical impulses to the stem cells in the hope of continuing the stem cells proliferation.   We also plan to use Netrin-1 protein 1) harness and study its effects to facilitate the stem cells to repair itself to and use to cause stem cells to proliferate 2) and cause the stem cells to convert into axons for spinal chord and brain injuries 3) we could also use it in conjunction stem cell injections.     Other Topical Ointments include:   Retin-A (tretinoin) perhaps in combination with hydroquinone and cortisone cream and as well alpha hydroxy acids and vitamin C serums and possibly steroids such as triamcinolone.  We are using Helix Aspera Muller Glycoconjugates snail/slug slime to enhance proliferation of stem cells. It is comprised complex sugar molecules bound to proteins, peptides, enzymes, coenzymes, oligoelements that play a role in messengers's communication between cells moderators of inflammatory reaction when skin if damaged, and regulators of fiberoblast proliferation and functions of the skin matrix and new connective tissues such as collagen and elastin fibers and water holding molecules (glycosaminoglycans and proteoglycans.     The Below is Taken BioSkincare Website:   FACTORS THAT HELP TO RECOVER THE NORMAL PHYSIOLOGY OF THE SKIN.  ~ Low molecular weight proteins that activate Fibroblast Growth Factor activity. Fibroblast proliferation is key for they are the stem cells in the basal membrane of the skin that give rise to new connective tissues: collagen and elastin fibers plus the glycosaminoglycans and proteoglycans that act as the glue that bind cells together and as fillers with a large capacity to withhold many times their weight in water and give skin strenght, elasticity and the capcity to withstand overstretching without breaking of skin fibers.   ~ High molecular weight proteins that work as Oxygen carriers (copper-haemocyanin) ~ Copper peptides that induce the degradation of "extra-large" collagen aggregates found in scar tissues such as those in stretch marks, and promotes the synthesis of smaller more regular collagen as is normal skin ~ Enzymes, both with Collagenase and Gelatinase activities that degrade damaged proteins and scar tissues ~ Hyaluronic Acid, provides deep tissue hydration ~ Glycoproteins / mucopolysaccharides that act as signaling molecules and enhance communication among and within cells, thus coordinating an orchestrated process that recognizes damaged and scar tissues, and activates the enzymes that "digest" or further degrade them into their amino-acid and other components, and uses those same components for the reconstruction of the extracellular matrix ~ Trace elements (Copper, Zinc, Iron, Calcium), some acting as coenzymes, necessary for Cellular Renewal, ~ Low molecular weight antioxidant compounds with Antioxidant & Anti-inflammatory effects.   To remove the scarring tissue from getting out of the way for the stem cells to bond to the healthy unscarred spinal cord tissue, in addition to substances such as Dermatix, we plan to try surgical procedures such as Dermabrasion (30% GP) and Chemical Peel (10%).   We also plan to use it in conjunction with (prior to) injecting stem cells into scarred injury (eg.  scarred tissue) anywhere where there is scarred tissue.     After removing the scarring, and adding stem cells to the injured area of the spinal chord, we use netrein-1 then we apply electrodes to prime the stem cells to grow into stem cells.   Additionally we could use detectors for faint muscle signals (initially with robotic help the limb's movement) then as the signal gets stronger, by retrains the brain signalling (neuron sounds or other form or mental activity detector) we could add neuron muscle patches that strengthens the muscle and eventually turn down the strength of the nerve patch signal and teach the patient to concentrate to increase the brains and nerve signals that operate the limb movement manually.   Furthermore, we will treat the stem cells with retinoic acid and vitamin A and (different concentrations of) frizzled WNT (G proteins), (sonic) hedgehog (agonists - Hh protein, Hh-Ag1.1, Hh-Ag1.2, vehicle (DMSO) pathways SAG, purmorphamine (smoothened) and nerve growth factor (NGF), brain derived neutrophic factor (BDNF), neutrophin-1 (NT-1), neutrophin-2 (NT-3), neutrophin-4 (NT-4) and possibly other growth factors. We also are looking at transcription factors Pax7, MNR2Nkx2.2. We culture stem cells in dibutyrl camp (dbcAMP), then we add rolipram, GDNF, and with motor neurons and neurons and different types of interneurons (as converted to stem cells by differing concentrations of sonic hedgehog). Dr. Kerr.   Breaks that need new bridging... the sciatic nerve extends from the spine down the back of the hind leg, and extending into the peripheral nervous system and all the way down to the gastronemius muscle in the lower leg and formed functional connections called synapses, with the muscles. Dr, Kerr.   We are also developing ways to observe cells innards via micro (real time x-ray, Cat Scan, fMRI, MRI or micro video probe - in REAL TIME 1) Lower Power to see through the cell membrane to see the cytoplasm and organelles eg mitochondria, 2) Medium Power to see through the mitochondria and 3) High Power to see through the nuclear membrane). The idea in this case is to sort the types of neural cells at a stem cells early stage. We are initially looking setting up an assembly line of colonies that the micro video camera observes to determine the different cells we look for types by shapes, size (of cells and organelles) positioning/orientation and placement (where suspended), conductivity (effects of electro pulse on different types of cells) of innards to their DNA orientation, make up of cytoplasm and percentage relative size of eg. cytosol to mitochondria (numbers), protrusions on the cellular membrane (receptors) - eg. dendrites are shown to have axons (but are they there in early stage of development...).   Then there is the question of do we need to separate the neural cells if the cells are used for spinal cord injury.   With regard to the spinal cord chemicals we plan to try vascular endothelial growth factor (VEGF) and insulinlike growth factor (IGF-1) also nicotinamide adenine dinucleotide (NAD)...   We will experiment with micro injection (piercing through to the nuclear membrane -with a very fine syringe, a nano or micro syringe) and/or electropulse/laser/fuse Oct3/4, Sox2, KIf4, c-Myc and Wnt (together with telomerase). Possibly after the colony of stem cells have stopped proliferating (from the nucleus removed eggs), to extend the amount of stem cells produced per batch; or the other way around, by starting out with reverse stem cells then after they have stopped proliferating, micro inject and/or electrofuse their DNA into a nucleus removed egg; thus alternating in case concentrating on just one method, which may concentrate the weaknesses of one alternative. We could also do both in the same round, that is, micro syringe and/or elctro/laser/fuse Oct3/4, Sox2, KIf4, c-Myc and Wnt together with telomerase into the nuclear membrane, after the electro shock to fuse the DNA material, we could then inject the DNA material in a nucleus removed egg. And electropulse to initiate division.   We could also add the above paragraph's process to the process of converting egg cells into stem cells.   That is to micro injection (piercing through to the nuclear membrane - with a very fine syringe, a nano or micro syringe) and/or electropulse/shock/laser/fuse Oct3/4, Sox2, KIf4, oncogene c-Myc and Wnt (together with telomerase) into an egg. Currently the bottle neck is proliferation because many medical uses require huge numbers of stem cells of which current technology cannot provide and there is the safety factor and a large pool of the adult's same stem cells need to last (keep alive) and then the next challenge is to differentiate into type of needed cells.   We could manipulate the genes when the egg derived stem cells stop proliferating.     And/or we could manipulate the genes of the eggs before they are converted into stem cells.   And/or we could manipulate the genes during the conversion of eggs to stem cells process.     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 could also combine all three eg. take a egg - from egg bank - from several females in case the success ratio for cloning is low; so the same female doesn't need to contribute so many eggs all by herself (remove its nucleus of the egg) and having multiple female donors when cloning success ratio is low also allows males to try in large numbers and then we add the manipulated genes to the egg or to the stem cells after proliferation... such as cases where even replating of colonies have stopped proliferating.   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.     See our past patent Application No.: 2,602,876, Owner: VOON, GERARD, Title:  TISSUE CULTURE AND CLONING THINGS...     Further in possibly in combination with the processes above, Techniques, Methods, Obtain fertilize eggs naturally, let it forma natural blastocyst, then remove the stem cells and remove their nucleus of the stem cells and add the adult DNA and electro shock the or laser or heat shock the cytoplasm and the DNA to fuse and divide into a blastocyst again and remove the stem cells and possibly using for example, micro injection (piercing through to the nuclear membrane - with a very fine syringe, a nano or micro syringe) and/or electropulse/shock/laser/fuse Oct3/4, Sox2, Klf4, oncogene c-Myc and Wnt (together with telomerase) into an the adult DNA impregnated stem cells. Currently the bottle neck is proliferation because many medical uses require huge numbers of stem cells of which current technology cannot provide and there is the safety factor and a large pool of the adult's same stem cells need to last (keep alive) and then the next challenge is to differentiate into type of needed cells. We are considering this method because something maybe missing without the sperm egg fertilization that creates immortal or at least human lifespan of stem cells that are found in natural fertilization.     We are also considering stem cells especially specialized cells for muscle augmentation and cosmetic surgery. We could grow fast twitch muscles (tissue cultured and electropulsed - to harden and tone the muscles, grown on scaffolds - to grow in the shape, distribution, size desired, - carved into shape before, we slit the skin (and/or flesh).     In addition to all the uses such as scarred dead tissue damaged (0.5%) blockage, stem cells could be centrifuged and mixed with a blood and/or blood substitute and multiple micro syringed. We could also use it for cosmetic cosmetic surgery eg. half lipo suction and half new stem cells added to cellulite (the new stem cells will increase muscle to fat ratio and burn more calories).