Stem Cells PDF

Nov. 12th ​ - ESC: Embryonic Stem Cells ​ - ASC: Adult Stem Cells ​ - iPSC: Induced Pluripotent Stem Cells ​ - Stem Cells are extraordinary because: o They can divide and make identical copies of themselves over and over again (Self-Renewal) o Remain Unspecialized with...

Nov. 12th ​ - ESC: Embryonic Stem Cells ​ - ASC: Adult Stem Cells ​ - iPSC: Induced Pluripotent Stem Cells ​ - Stem Cells are extraordinary because: o They can divide and make identical copies of themselves over and over again (Self-Renewal) o Remain Unspecialized with no ‘specific’ function or become.... o Specialized (Differentiated) w/ the potential to produce over 200 different types of cells in the body. ​ - Stem cell may differentiate or remain a stem cell ​ - Pluripotent stem cells can differentiate into every cell type in body Stem cell Description Examples type Totipotent Each cell can develop into a new Cells from early (1-3 days) individual embryos Pluripoten Cells can form any (over 200) cell types Some cells of blastocyst (5 to t 14 days) Multipoten Cells differentiated, but can form a Fetal tissue, cord blood, and t number of other tissues adult stem cells  ​ - Stem cells can be classified into three broad categories, based on their ability to differentiate. ​ - Totipotent stem cells are found only in early embryos. Each cell can form a complete organism. ​ - Pluripotent stem cells exist in the undifferentiated inner cell mass of the blastocyst and can form any of the over 200 different cell types found in the body. ​ - Multipotent stem cells are derived from fetal tissue, cord blood and adult stem cells. Although their ability to differentiate is more limited than pluripotent stem cells, they already have a track record of success in cell-based therapies. Here is a current list of the sources of stem cells: o Embryonic stem cells - are harvested from the inner cell mass of the blastocyst seven to ten days after fertilization. o Fetal stem cells - are taken from the germline tissues that will make up the gonads of aborted fetuses. Downloaded by Casey Garnett ([email protected]) lOMoARcPSD|36642292 o Umbilical cord stem cells - Umbilical cord blood contains stem cells similar to those found in bone marrow. o Placenta derived stem cells - up to ten times as many stem cells can be harvested from a placenta as from cord blood. o Adult stem cells - Many adult tissues contain stem cells that can be isolated. ​ - The human body has approximately 200 different cell types. ​ - The potential uses of stem cells in regenerative medicine are huge. ​ - Why is Stem Cell Research So Important to All of Us? o Stem cells allow us to study how organisms grow and develop over time. o Stem cells can replace diseased or damaged cells that can not heal or renew themselves. o We can test different substances (drugs and chemicals) on stem cells. o We can get a better understanding of our “genetic machinery.” ​ - While it is possible to get pluripotent stem cells from aborted fetuses, this has not been done since the 1990s.  ​ - Nearly all Human Embryonic Stem Cells (HESC) in use today were derived from donated blastocysts, not aborted fetuses. ​ - Lab-grown fetal cell lines that are genetically related to cells collected from an aborted fetus decades ago have been used to test the safety and effectiveness of several common medicines and vaccines, including the mRNA vaccines made by Pfizer and Moderna ​ - Stages of Embryogenesis: o The early stages of embryogenesis are the point at which embryonic stem cell lines are derived. o The fertilized egg (day 1) undergoes cell division to form a 2-cell embryo, followed by 4-cell, etc. until a ball of cells is formed by the fourth day. o The ball becomes hollow, forming the blastocyst. o This is the stage at which pluripotent embryonic stem cell lines are generated. Following the blastocyst stage, the tissues of the embryo start to form and the cells become multipotent. o By day 5 the cells of the blastocyst have differentiated into the outer trophoblast (which give rise to the placenta) and the Inner Mass Cells (which give rise to the embryo). The blastocyst is a structure formed in the early development of mammals. Downloaded by Casey Garnett ([email protected]) It possesses an inner cell mass (ICM) which subsequently forms the embryo. The outer layer of the blastocyst consists of cells collectively called the trophoblast. This layer surrounds the inner cell mass and a fluid-filled cavity known as the blastocoele. The trophoblast gives rise to the placenta. lOMoARcPSD|36642292 ​ - The inner cell mass (the part that would form the fetus) of the embryo is isolated and disrupted to form embryonic cell lines. ​ - The outer trophoblast cells are discarded. This process destroys the embryo. ​ - Under special culture conditions, the cells of the embryonic lines can be cultured and then coaxed to form certain kinds of differentiated cell types.  ​ - In theory, these differentiated cells could be used to repair or replace defective cells or tissues. ​ - The source of blastocysts for hESC are nonviable embryos that were created for in vitro fertilization (IVF). ​ - Since many embryos fail to develop normally, clinics try to create at least twenty embryos to produce a single healthy child. ​ - The unused embryos are either destroyed or donated for medical use. ​ - Non-viable embryos classified by morphological criteria under brightfield optics. -------------------- o (A.) Embryo showing pronounced signs of degeneration o (B.) Single-celled embryo o (C.) Embryo with three large blastomeres o (D.) Embryo with at least one large blastomere (occupying 25–50% of embryo) as well as multiple smaller blastomeres or cytoplasmic fragments o (E.) Embryo with multiple small blastomeres and/or cytoplasmic fragments o (F.) Embryo showing signs of compaction o (G.) Embryo with one or more blastomeres with a cavity o (H.) Embryo with a central cavity and an abnormal inner cell mass ​ - The number of defective, non-viable, embryos surprises people, but in fact this is natural and not a result of IVF. ​ - Most human embryos that are produced in the natural way (i.e. mating) are also defective. o They do not thrive in the woman and are shed with her next menstrual cycle. Downloaded by Casey Garnett ([email protected]) o oo This single nucleus can then be tagged for chromosomal markers. Specific chromosomes can be tagged with fluorescent markers. In this case there are two each of green, red, yellow and purple, but three light blue dots, indicating an abnormal number (three) of chromosomes that have that particular marked gene. lOMoARcPSD|36642292 ​ - Cells from 3-day old embryo showing a chromosomal trisomy genetic defect (blue dots). ​ - Blastocysts so obtained provide a route to embryonic stem cells from otherwise nonviable embryos ​ - A single cell can be removed from the 2-3 day old embryo without affecting the viability of the remaining cells. ​ - A chromosomal nondisjunction event can result in a gamete with an extra chromosome #21 which results in Down Syndrome. ​ - Trisomies are caused by a chromosomal nondisjunction event that occurs during gametogenesis. It can occur in either the sperm or the egg cell. ​ - A chromosomal trisomy is characterized by the presence of three copies of a chromosome rather than the normal two copies per nucleus. ​ - Trisomy 13 (Patau Syndrome) isn't always fatal. o But doctors can't predict how long a baby might live if they don't have any immediate life-threatening problems. o However, babies born with trisomy 13 rarely live into their teens. ​ - Most babies with trisomy 18 (Edwards Syndrome) die before they are born. o The majority of those who make it to term die within five to 15 days, usually due to severe heart and lung defects. ​ - Trisomy 18 occurs in about one out of every 6,000 to 8,000 live births and trisomy 13 occurs in about one out of every 8,000 to 12,000 live births. ​ - What percentage of pregnancies go to full term? o Studies have found that 30 to 50 percent of fertilized eggs are lost before or during the process of implantation. o About 10 to 20 percent of known pregnancies end in miscarriage, and more than 80 percent of these losses happen before 12 weeks. o Between 50 and 70 percent of first-trimester miscarriages are thought to be random events caused by chromosomal abnormalities in the fertilized egg. - Life begins at conception. o But conception only rarely leads to life. o 30% make it all the way to full term birth Downloaded by Casey Garnett ([email protected]) lOMoARcPSD|36642292 - February 16, 2024: Alabama’s supreme court ruled embryos are ‘extrauterine children’. o In considering a “wrongful death” lawsuit between an Alabama fertility clinic and hospital and three couples undergoing fertility treatments. o Several frozen embryos belonging to the couples were accidentally destroyed in a hospital cryonursery. o The Alabama Supreme Court ruled that frozen embryos conceived via in vitro fertilization (IVF) are considered “children” under the state’s Wrongful Death of a Minor Act. o The accident was caused by a hospital patient who went into the cryonursery and removed some of the frozen embryos. o For reasons that aren't clear to doctors, any fetus Gabby Goidel (a 26-year-old property manager in Auburn, AL) carries has a higher-than-average likelihood of genetic abnormalities, so there is a slim chance she'd be able to carry a pregnancy to term without in-vitro fertilization. o According to the decision, people in Alabama could be sued for intentionally, or even accidently, destroying a frozen embryo, raising questions about in-vitro fertilization. ​ - February 21, 2024: The University of Alabama at Birmingham health system has paused in vitro fertilization procedures following an Alabama Supreme Court decision due to fear of criminal prosecution and lawsuits, a spokeswoman said. ​ - In 2007 James Thomson, who was the first to establish a line of hESCs, achieved another first when he was able to “reprogram” adult skin stem cells into induced pluripotent stem cells (iPSC). ​ - 2012- Shinya Yamanaka, and John Gurdon shared the Nobel Prize in Physiology or Medicine for their work with transforming adult skin stem cells into iPSCs. ​ - It's now possible to grow stem cells in a lab, not just from embryonic tissue but also by turning back the clock on an already developed cell like one from the  skin, bypassing the embryo altogether with four important fountain-of- youth genes that rework the skin cell's DNA machinery and make it stemlike again ​ - The reprogramming of adult cells into iPSCs involves the use of retroviruses to genetically alter a cell’s DNA and thus limits their use in therapy. ​ - Induced pluripotent stem cells (also known as iPS cells or iPSCs) are a type of pluripotent stem cell that can be generated directly from adult cells. o The iPSC technology was pioneered by Shinya Yamanaka’s lab in Kyoto, Japan, who showed in 2006 that the introduction of four specific genes encoding transcription factors could convert adult cells into pluripotent stem cells. Downloaded by Casey Garnett ([email protected]) lOMoARcPSD|36642292 o He was awarded the 2012 Nobel Prize along with Sir John Gurdon "for the discovery that mature cells can be reprogrammed to become pluripotent." ​ - The reprogramming factors can lead to the turning on of oncogenes (c- Myc) associated with rapid cell proliferation ​ - iPSCs are typically derived by introducing products of specific set of pluripotency-associated genes, or “reprogramming factors”, into a given cell type. ​ - The original set of reprogramming factors (also dubbed Yamanaka factors) are the transcription factors Oct4 (Pou5f1), Sox2, cMyc, and Klf4. o While this combination is most conventional in producing iPSCs, each of the factors can be functionally replaced by related transcription factors, miRNAs, small molecules, or even non- related genes such as lineage specifiers. ​ - Myc (c-Myc) is a regulator gene that codes for a transcription factor. o The protein encoded by this gene is a multifunctional, nuclear phosphoprotein that plays a role in cell cycle progression, apoptosis and cellular transformation. ​ - Problems with iPSCs: o Very low transformation rates (0.01% to 0.0001%) o One of the four genes (Myc) is an oncogene that can cause cancer. o Gene inserts can disrupt normal cell function and induce mutations and epigenetic changes. o iPSC are not easily transplanted and may elicit an immune response, even if returned to the same individual from which they were derived. o iPSCs do not easily make tissues derived from mesoderm (e.g. liver, pancreas, etc.). ​ - Another problem with using iPSC in treating diseases caused by genetic abnormalities (e.g. MS, diabetes, leukemia, etc.) is that a patient’s own stem cells carry the same genetic defects that led to the disease in the first place. ​ - Advantages and Disadvantages of Embryonic and Adult Stem Cells Embryonic S.C. Adult S.C. “Pluripotent” (can become any cell) “Multipotent” (“can become many but not any”) Stable. Can undergo many cell Less Stable. Capacity for self- renewal is divisions. limited. Easy to obtain but blastocyst is DifÏcult to isolate in adult tissue. destroyed. Possibility of rejection?? Host rejection minimized Downloaded by Casey Garnett ([email protected]) lOMoARcPSD|36642292 ​ - Direct cell reprogramming, also called transdifferentiation, allows for the reprogramming of one somatic cell type directly into another, without the need to transition through an induced pluripotent state. o Thus, it is an attractive approach to develop novel tissue engineering applications to treat diseases and injuries where there is a shortage of proliferating cells for tissue repair. ​ - ViaCyte is a regenerative medicine company focused on delivering novel stem cell-derived cell replacement therapies as a functional cure for all type 1 diabetes and a next-generation treatment for insulin- requiring type 2 diabetes  ​ - ViaCyte is the only company with stem cell-derived islet replacement candidates undergoing clinical testing and the only group to show that the implanted cells are capable of producing insulin in people with type 1 diabetes. o The Company’s promising candidates have the potential to deliver a functional cure for people with type 1 diabetes and a significant advancement for people with insulin-requiring type 2 diabetes. ​ - To create a Pancreatic β cell from a hESC precursor one needs... o 1. The ability to synthesize insulin into granules. o 2. A GLUT-2 receptor in the plasma membrane o 3. Expression of Glucokinase in the cytoplasm o 4. An ATP-sensitive potassium ion pump in the plasma membrane o 5. A Voltage-gated calcium ion channel o 6. The ability to release insulin into the extracellular space ​ - Let us look at the "glucose sensor" system first. o o o o oo The pancreatic beta cell's primary function is to correlate release of insulin with changes in blood glucose concentration. Obviously, these cells must have a sensitive glucose-measuring device. Nature has achieved this by equipping the beta cell with a glucose transport protein (GLUT2) and a kinase (glucokinase) both of which have low afÏnities for glucose. Glucose-6 Phosphate (G6P) is the intracellular form of blood glucose. Following tissue uptake of glucose, via facilitated diffusion (Thorens 1993), phosphorylation traps the glucose by placing a negative charge on the molecule, preventing its diffusion back across the cell membrane into the blood. GLUT2 is quite active, but the Km for glucose is around 5 mmol/l. Therefore, transport of glucose into the beta cell is rapid, but only when the blood glucose concentration exceeds post-meal levels. - This triggers the pumping out of the cell of positively charged potassium ions and thus the voltage difference between the inside and outside of the cell (-60 mV) is altered and at a certain threshold Downloaded by Casey Garnett ([email protected]) lOMoARcPSD|36642292 will open up voltage gated calcium ion channels and calcium ions will flow into the cell. ​ - The uptake of calcium ions in the cytoplasm causes the insulin granules to fuse to the cell membrane and release insulin into the blood. ​ - Insulin loaded vesicles are already premade and waiting in the beta cells, it is only their release that is triggered by high blood sugar so the system responds very quickly once the threshold of 5.5 nmol/l of glucose is exceeded. ​ - When modified STEM cells that can release insulin in response to high blood sugar levels were transplanted into diabetic mice they gained the ability to better regulate their blood sugar levels. ​ - These stem-cell-derived β cells (SC-β) express markers found in mature β cells, flux Ca2+ in response to glucose, package insulin into secretory granules, and secrete quantities of insulin comparable to adult β cells in response to multiple sequential glucose challenges in vitro ​ - Pluripotent stem cells seem to be the best option for clinical applications of β-cell regeneration in the near future, as these cells have been demonstrated to represent an unlimited source of functional β cells ​ - Pros of ViaCyte Treatment: o Cells derived from ESCs can respond to changes in blood sugar levels and produce insulin at appropriate times. o ESCs are protected from the patient’s immune response by a thin permeable membrane that allows for the transfer of vital nutrients, proteins, oxygen, CO2, glucose, insulin, and other hormones. - Cons of ViaCyte Treatment: o The insulin secreting ESCs are not in direct contact with the patient’s vascular system and the thin permeable membrane limits the transfer of vital nutrients, proteins, oxygen, CO2, glucose, insulin, and other hormones. o This limits the volume of the ESCs as all molecules must move by diffusion.  ​ - ViaCyte is developing the PEC-Encap product candidate as a functional cure for type 1 diabetes. ​ - For PEC-Encap, the pouch is designed to fully contain the implanted cells but still allow vital nutrients and proteins, such as oxygen, glucose, insulin, and other hormones, to travel between the cells inside the device and the blood vessels, which grow along the outside of the device. ​ - This device is also designed to prevent immune cells from directly contacting the implanted cells so they may thrive and function without provoking an immune response or being destroyed. Downloaded by Casey Garnett ([email protected]) lOMoARcPSD|36642292 ​ - In the case of PEC-Encap, the Encaptra® system has generally prevented immune rejection and immune sensitization and effectively protects implanted cells from the patient’s adaptive immune system. ​ - Approximately 1.25 million people in the United States have type 1 diabetes (statistics from JDRF). Worldwide, an estimated 42 million people have type 1 diabetes (~10% of 420 million cases of all forms of diabetes, IDF). ​ - The biologically active component of the PEC-Encap product candidate is stem cell-derived pancreatic islet cell progenitors, called PEC-01TM cells. ​ - These cells are contained within the Encaptra System and implanted in a patient subcutaneously. ViaCyte has shown that once implanted and successfully engrafted, the PEC-01 cells mature into beta cells that secrete insulin in a regulated manner to control blood glucose levels. ​ - Other cells of the normal human islet are also produced. ​ - The Encaptra System serves to protect the PEC-01 cells from the host immune system thus eliminating the need for immune suppression drugs commonly used with other transplants. ​ - A critical enabling component of the Encaptra System that contains and protects the cells is a specifically designed semi-permeable membrane that encapsulates the cells but allows for diffusion of oxygen and nutrients into the device and insulin and other hormones out of the device and into the circulation. ​ - This membrane enables the formation of a vascular network on the surface of the device after implantation. ​ - The Encaptra System membrane is composed of an expanded PTFE composite, similar to materials that are used in Gore’s implantable medical products. ​ - The goal of the collaboration with Gore is to engineer and deliver materials and device designs that facilitate the vascularization by modifying the host foreign  body response to the Encaptra System and maximize the engraftment of the PEC-01 cells. ​ - Fibrosis is the formation of excess fibrous connective tissue (scar tissue) in an organ or tissue in a reparative or reactive process. ​ - The immune system often builds up a wall of dense scar tissue around implanted medical devices, a process known as fibrosis. o Eventually enough collagenous material surrounds the device that it greatly limits diffusion. ​ - In clinical trials the PEC-Encap has run into some problems in that scar tissue forms around the implant and thus limits the ability of signal to diffuse in (i.e. blood sugar levels) and for insulin to diffuse out. ​ - The Stice lab has successfully transformed stem cells into nerve cells that function in a normal way. o The technique holds promise for those who suffer from degenerative diseases such Parkinson’s, Alzheimer’s and Multiple Sclerosis and neural injuries of spinal cord and head trauma. Downloaded by Casey Garnett ([email protected]) lOMoARcPSD|36642292 ​ - 2001- President George W. Bush restricts federal funding for embryonic stem cell research to 21 existing cell lines. ​ - 2009- President Barack Obama reinstates federal funding for embryonic stem cell research. ​ - The Company’s new name also provides consolidation. o The Company is the result of a three-way merger in 2004 of Novocell, Inc. (Irvine, CA), Cythera, Inc. (San Diego, CA) and BresaGen, Inc. (Athens, GA). ​ - The use of Human Embryonic Stem Cells in biomedical research remains highly controversial. ​ - A number of years ago some representatives in the Georgia General Assembly suggested legislation that would ban research using hESCs in Georgia. ​ - In contrast, California actually offered financial incentives to such researchers. ​ - So in deciding where to invest millions of dollars to develop their products, ViaCyte moved to California. o The facility in Athens still carries out some research but is prepared to move out of Georgia should the political situation change.  ​ - Stem cells from bone marrow form a number of cell types of the immune and circulatory system. o These stem cells have been used to cure diseases since the 1960's. ​ - Erythropoiesis (from Greek 'erythro' meaning "red" and 'poiesis' meaning "to make") is the process which produces red blood cells (erythrocytes). ​ - White blood cells (WBCs), also called leukocytes are the cells of the immune system that are involved in protecting the body against both infectious disease and foreign invaders. ​ - All white blood cells are produced and derived from multipotent cells in the bone marrow known as hematopoietic stem cells (HSC). ​ - Leukocytes are found throughout the body, including the blood and lymphatic system. ​ - Conditions that can be treated with BMSC transplantation: o Acute and chronic leukemias o Sickle Cell Disease o Aplastic anemia o Congenital immunodeficiency diseases o Lymphomas o Metabolic disease of childhood o Myelodisplasia o Thalassemia ​ - Patients with acute myeloid or lymphoblastic leukemia may benefit from BMT. Downloaded by Casey Garnett ([email protected]) lOMoARcPSD|36642292 ​ - Patients with acute myeloid leukemia transplanted in first remission can now expect an approximately 50 to 60% likelihood of long-term disease-free survival. ​ - Similar probabilities are also achievable after transplantation of adults with acute lymphoblastic leukemia in first remissions. ​ - Probability of relapse correlates with remission status at the time of the transplant, ranging from 20% in first remission to 60% with more advanced disease. ​ - Long-term survival for patients with chronic myelocytic leukemia who receive BMT in the phase of remission is 60 to 70%. ​ - Pediatric BMT has expanded because of its potential for curing children with genetic diseases (eg, thalassemia, sickle cell anemia, immunodeficiencies, inborn errors of metabolism). ​ - To minimize rejection the donor and recipient should have nearly identical gene variants for Human Leukocyte Antigen (HLA) complex.  ​ - There are 309 known alleles of HLA-A, 167 of HLA-C, and 563 different alleles of HLA-D. ​ - If the alleles sorted completely separately these three HLAs alone could form 29,052,449 different combinations (309 X 167 X 563 = 29,052,449_ but since these genes are close together on the same chromosome they are typically inherited as a unit (in other words no crossing over occurs within the HLA complex) this makes actual odds of finding a matching donor is about 1:100,000. ​ - The key limiting factor in the use of BMT is the lack of donors. ​ - Because only 25 to 30% of patients have an HLA-identical sibling, alternative donors are often required. ​ - Two possibilities exist: o o o (1) Marrow can be procured from unrelated living donors; marrow donation is a simple, safe procedure. National and international registries of prospective volunteer donors are being expanded to increase the likelihood of finding an exact HLA match for any given recipient. (2) Related donors who are not HLA-identical have been used with increasing frequency. Results with either procedure suggest long-term disease-free survival probabilities of 30 to 50% in patients with acute and chronic leukemia or aplastic anemia; ie, in most situations the results are somewhat inferior to those with marrow from HLA- identical siblings. ​ - Another option for BMT is autologous transplantation (removal of a patient's own marrow when a complete remission has been induced, followed by ablative treatment of the patient with the hope of destruction of any residual tumor and rescue with the patient's own bone marrow). ​ - Since an autograft is used, no immunosuppression is necessary other than the short-term high-dose chemotherapy used for tumor Downloaded by Casey Garnett ([email protected]) lOMoARcPSD|36642292 eradication and bone marrow ablation; posttransplant problems with GVHD are minimal.  ​ - Indications for autologous BMT are relapsed, chemotherapy-sensitive lymphoma, in which a 30 to 40% success rate has been achieved, and acute leukemia in remission, in which 20 to 50% success rates have been observed. ​ - Success rates are inferior with more advanced disease and with responsive solid cancers (eg, breast or germ cell tumors). ​ - Two major obstacles remain for successful application of autologous BMT: o the possibility of contamination of the marrow inoculum with tumor cells, and the absence of graft-vs.-tumor activity (in contrast with that seen in allogeneic BMT), both of which contribute to the observed higher rates of tumor recurrence. ​ - Thus, developing schemes for ex vivo marrow purging and for recipient immune modulation posttransplant is an active area of research. ​ - In December 2010, twenty-four year old Sonia Rai was diagnosed with Acute Myelogenous Leukemia. o Her brother Sumit dropped everything to be with her in Boston and to organize a nationwide search for a potential bone marrow donor who could save Sonia. o Among her last wishes was that the efforts that went into the Cure Sonia campaign continue and grow in the hope that other lives will be saved. ​ - Among the 7 million registered donors currently, only 2% are South Asians. ​ - Ethnic minorities have only a 30-40% chance of finding a match from the existing NMDP registry. ​ - Caucasians currently have an 80% chance of finding a match. ​ - While the odds of finding a random HLA match might be about 1:100,000 the odds among genetic siblings (same parents) is 1:4. o This is because the HLA complex is typically inherited as a unit. Sadly for Sonia, Sumit was not a good match. ​ - Hematopoietic stem cells (HSCs) develop into blood cells ​ - The key to treating individuals with diabetes depends on getting insulin to be released into the blood at the appropriate levels and at the appropriate times. ​ - With only three minutes of exposure to music (delivered by having the mice lying on top of a loudspeaker) resulted in a substantial release of insulin from the engineered cells. ​ - Insulin release depends on the opening of voltage gated ion channels allowing Ca2+ to enter the cell. ​ - In the case of pancreatic beta cells this is triggered with a change in potassium (K) channels opening. Downloaded by Casey Garnett ([email protected]) lOMoARcPSD|36642292 ​ - Once Calcium ions enter the cell the vesicles containing insulin will fuse with the plasma membrane and release their contents into the extracellular environment and ultimately into the blood stream. ​ - Making a stem cell that has all of these functions is difÏcult. What if one could simplify it by just having two components, an inducible calcium ion channel and the ability to synthesize insulin granules? ​ - Mechanical stimuli, e.g., compression, membrane tension, poking, shear stress, stretching, or suction activates calcium influx through mechanosensitive ion channels. ​ - Then, calcium acts as a signal molecule on various pathways which may lead to, e.g., cell proliferation [43,44,45,46]. ​ - Two models of mechanosensitive ion channel gating have been established. o (A) In the force-from-lipids model, alterations in lipid organization lead to channel activation; o (B) In the force-from-filaments model, the causative factors are changes in the extracellular matrix and cytoskeleton ​ - The design of our MUSIC cellular device involves rewiring the intracellular calcium surge actuated by calcium-permeable mechanosensitive channels in response to music to drive immediate calcium-triggered vesicular release of biopharmaceuticals ​ - When vibrations deformed the membrane, the mechanosensitive ion channel allowed Ca2+ ions to enter the cell and trigger the fusion of insulin containing vesicles. ​ - Music with a driving base beat are more likely to trigger the influx of calcium ions and thus release insulin. Nov. 14th ​ - Glucose comes in as starch, glycogen, or simple and complex sugars. It is either used or stored as glycogen, mostly in the liver. ​ - The term saccharide refers to the unit structure of carbohydrates. ​ - Carbohydrates are simple organic compounds that are aldehydes or ketones with many hydroxyl groups added usually on each carbon atom not part of the aldehyde or ketone functional group. ​ - The general chemical formula of carbohydrates is Cn (H2O) n. o Not all carbohydrates follow this formula and are slightly different in structure from this rule ​ - Glucose is the main source of metabolic energy in living things as it serves as the main substrate in glycolysis* ​ - Glycolysis is a cytoplasmic pathway which breaks down glucose into two three-carbon compounds and generates energy.  ​ - Glucose is trapped by phosphorylation, with the help of the enzyme hexokinase. ​ - Adenosine triphosphate (ATP) is used in this reaction and the product, glucose-6-P, inhibits hexokinase. Downloaded by Casey Garnett ([email protected]) lOMoARcPSD|36642292 ​ - Glycolysis takes place in 10 steps, five of which are in the preparatory phase and five are in the pay-off phase. ​ - Phosphofructokinase is the rate-limiting enzyme. ​ - ATP is generated by substrate-level phosphorylation by high-energy compounds, such as 1,3-bisphosphoglycerate and phosphoenolpyruvate. ​ - Glycolysis is used by all cells in the body for energy generation. ​ - The final product of glycolysis is pyruvate in aerobic settings and lactate in anaerobic conditions. ​ - In many organisms Pyruvate enters the Krebs cycle for further energy production. ​ - All known organisms, from bacteria to humans, are able to metabolize glucose. ​ - Glucose is the universal energy molecule used by all known forms of life on Earth. ​ - Larger number of sugar molecules can join each other to form large polysaccharides such as starch, the storage product of plants and green algae. Starch is a branched α 1-4 linked glucan (~25% amylase + ~75% amylopectin). o The designation “1-4” branching refers to which carbon atoms in the glucose molecule are involved in forming the bridges in the primary linked molecule. ​ - Glycogen is made of linked glucose molecules and is a major storage product of animal cells, often found in high abundance in the liver. o Like starch it is a branched α 1-4 linked glucan with the branches being α 1-6 bond off the stem. o branching off by α(1→6) glycosidic bonds between the first glucose of the new branch and a glucose on the stem chain ​ - Cellulose is a β 1-4 linked glucan that forms unbranched linear molecules that are held together by hydrogen bonds. ​ - Cellulose is an unbranched, linear molecule.  ​ - The fact that these linear polymers can line up side by side and connect to one another via hydrogen bonding makes it very difÏcult for an enzyme (cellulase) to gain access to the bonds and break cellulose back into glucose monomers. ​ - Chitin is a long-chain polymer of a N-acetylglucosamine, a derivative of glucose, and it is found in many places throughout the natural world. o It is the main component of the cell walls of fungi and the exoskeletons of insects and crustaceans. o Like cellulose, the carbohydrate chitin forms long, linear, unbranched molecules. ​ - Carbohydrates or Amylase enzymes break down starch into sugar ​ - The branched nature of starch (and glycogen) molecules allows the enzymes to gain access to the bonds that link the glucose monomers and to break them apart. Downloaded by Casey Garnett ([email protected]) lOMoARcPSD|36642292 - A typical blood sugar level in humans is about 90 mg/dL but in it can vary by as much as 50% over the course of the day. o Blood glucose is measured in milligrams/deciliter (mg/dL). o To put what follows in context, you can think of 100 mg/dL as just a few grains of sugar in a half a cup of water. o Assuming that you’re not a diabetic or prediabetic (much more about those conditions below), your set point for blood glucose is probably about 90 mg/dL (indicated by the dotted line at “E”). o When you eat, particularly if it’s a meal with starch or sugar, then your blood glucose will rise to a higher level: up to about 140 mg/dL (point “B”). o At that point, homeostatic mechanisms will kick in, lowering your blood glucose back to its set point. o If you go a few more hours without eating, then your glucose level will continue to fall. o At a certain point (“C”) other homeostatic mechanisms will kick in to raise your blood sugar back to the set point. o Your blood sugar will continue to oscillate around a set point until you eat again. ​ - Blood sugar levels are primarily maintained through the interactions of three organ systems; Intestines, Liver and Pancreas. ​ - In response to blood sugar levels that are either too high or too low the pancreas will secrete one of two different hormones; insulin and glucagon. o These hormones travel through the blood stream until they elicit a response in either liver or tissue cells. ​ - The human body maintains blood glucose (blood sugar) in a very narrow range. Insulin and glucagon are the hormones which make this happen. Both insulin and glucagon are secreted from the pancreas, and thus are referred to as pancreatic endocrine hormones. The pancreas serves as the central player in this scheme. o It is the production of insulin and glucagon by the pancreas which ultimately determines if a patient has diabetes, hypoglycemia, or some other sugar problem. ​ - What type of feedback loops control blood sugar levels? o Negative feedback loop the system works to move the blood sugar level in the direction that is opposite of the controlling condition and towards the set point. A positive feedback loop would make a high blood sugar level higher or a low blood sugar level lower. ​ - The human body maintains blood glucose (blood sugar) in a very narrow range. Insulin and glucagon are the hormones which make this happen. Downloaded by Casey Garnett ([email protected]) lOMoARcPSD|36642292 ​ - Both insulin and glucagon are secreted from the pancreas, and thus are referred to as pancreatic endocrine hormones. ​ - The pancreas serves as the central player in this scheme. ​ - It is the production of insulin and glucagon by the pancreas which ultimately determines if a patient has diabetes, hypoglycemia, or some other sugar problem. ​ - Insulin is a hormone that is secreted by the beta cells in the pancreas.  ​ - Insulin facilitates the movement of glucose into the muscle, fat cells, and liver cells. ​ - The pancreas is a glandular organ in the digestive system and endocrine system of vertebrates. I o n humans, it is located in the abdominal cavity behind the stomach. o It is an endocrine gland producing several important hormones, including insulin, glucagon, somatostatin, and pancreatic polypeptide which circulate in the blood. o The pancreas is also a digestive organ, secreting pancreatic juice containing bicarbonate to neutralize acidity of chyme moving in from the stomach, as well as digestive enzymes that assist digestion and absorption of nutrients in the small intestine. o These enzymes help to further break down the carbohydrates, proteins, and lipids in the chyme. o The pancreas is also known as a mixed gland. ​ - The peptide hormone insulin triggers movement of glucose across the cell membrane in an indirect manner known as an activation cascade. ​ - Effect of insulin on glucose uptake and metabolism: o Insulin binds to the membrane embeded insulin receptor ​ (1) which in turn starts many protein activation cascades (2). ​ These include: translocation of the Glucose transporter- 4 (Glut-4) to the plasma membrane and influx of glucose (3), Glycogen synthesis (4), Glycolysis in which glucose is reduced to pyruvate (5) and fatty acid synthesis (6). ​ - In its simplest form, an activation cascade comprises a set of components (typically proteins) that become sequentially activated in response to an external stimulus. ​ - In addition to the liver cells many tissues in the body, including muscle, are activated by insulin to take up glucose and use it in a wide variety of metabolic ways. ​ - Glut-4 transporters are found in liver cells, muscle cells and fat cells (both brown and white fat cells).  ​ - Insulin can up regulate certain cellular processes (e.g. glycolysis and glycogen synthesis) and down regulate others (e.g. gluconeogenesis). Downloaded by Casey Garnett ([email protected]) lOMoARcPSD|36642292 - In the homeostatic control of blood glucose levels, which component acts as an “effector”? o Liver Cells and Glucose trnsporter-4 o The liver can be an effector by either pulling glucose out of the blood (and concerting it to glycogen) or by converting glycogen into glucose and releasing it into the blood. o The Glut-4 receptor is an effector by facilitating the uptake of glucose from the blood and bringing it into cells, thus lowering the blood sugar levels. ​ - The pancreas both monitors blood sugar levels and produces a signal after comparing the levels against a set point ​ - The liver plays a major role in glucose homeostasis as the effector. ​ - When stimulated by insulin it will convert blood glucose into the storage carbohydrate glycogen. ​ - When stimulated by glucagon the liver will break down glycogen into glucose molecules and release then into the blood. ​ - Glycogen is the primary carbohydrate storage molecule in animals. The liver can act as an effector in either raising blood sugar levels or by lowering them. ​ - The sensors in the system are the GLUT-2 glucose channel on the pancreatic beta cells and the GLUT-1 glucose channels on the pancreatic alpha cells. ​ - The relative sensitivity of these two channels to blood sugar levels is what created the set point for the system. ​ - The cells of the pancreas function as both the receptor and the control center. ​ - Alpha cells sense when blood sugar levels have dropped too low and release glucagon. ​ - Beta cells sense when blood sugar levels are too high and release insulin.  ​ - The influx of glucose via the GLUT-2 receptor (1) triggers the production of ATP via metabolism (2 & 3) which closes the potassium channel (4) which stops the flow of positively charged potassium ions which raises the charge inside the cell relative to the outside of the cell which depolarizes the cell membrane (5) which causes the voltage- gated calcium ion channel (6) to open which causes the influx of calcium ions (7) from the outside which triggers the fusion of insulin containing vesicles to the plasma membrane (8) and the release of insulin (9)! ​ - After a carbohydrate-rich meal, glucose will diffuse into the blood from the small intestine. As a polar, water soluble molecule, glucose needs a channel to pass from the extracellular fluid (“A”) through the membrane (“B”) and into the cytoplasm (“C”). That channel is called GLUT2 (see “1”) and it allows glucose to enter the cytoplasm of the pancreatic Beta cells by facilitated diffusion. Once in the cytoplasm, glucose becomes the initial substrate for cellular respiration (“2”), Downloaded by Casey Garnett ([email protected]) lOMoARcPSD|36642292 resulting in increased ATP (“3”). This ATP binds with a potassium channel (“4”), causing it to close. With these positively charged potassium ions no longer diffusing out of the cell, the membrane becomes partially depolarized (“5”). ​ - Depending on where you are in your curriculum, what happens next might remind of how neurotransmitters are released from axonal bulbs, or about how muscle contraction occurs. Depolarization causes voltage dependent calcium channels (VDCCs, at “6”) to open. This allows calcium ions to diffuse into the cytoplasm. where they interact with insulin containing vesicles in such a way that induces exocytosis (“8”) releasing insulin into the bloodstream (“9”). ​ - Voltage-gated calcium channels (VGCCs), also known as voltage- dependent calcium channels (VDCCs), are a group of voltage-gated ion channels found in the membrane of excitable cells (e.g., muscle, glial cells, neurons, etc.) with a permeability to the calcium ion Ca2+. These channels are slightly permeable to sodium ions, so they are also called Ca2+-Na+ channels, but their permeability to calcium is about 1000-fold greater than to sodium under normal physiological conditions. ​ - At physiologic or resting membrane potential, VGCCs are normally closed. They are activated (i.e.: opened) at depolarized membrane potentials and this is the source of the "voltage-gated" epithet. The concentration of calcium (Ca2+ ions) is normally several thousand times higher outside the cell than inside. Activation of particular VGCCs allows a Ca2+ influx into the cell, which, depending  on the cell type, results in activation of calcium-sensitive potassium channels, muscular contraction, excitation of neurons, up-regulation of gene expression, or release of hormones or neurotransmitters. ​ - Control of glucagon secretion is not as well understood as that of insulin. Secretion of glucagon is clearly linked to the alpha cell's metabolism. Lack of substrate, anoxia and metabolic poisons lead to release of glucagon from these cells. In short, they release their hormone in response to "metabolic stress". As is the case of the beta cell's release of insulin, it has become clear that regulation of the membrane potential is decisive for control of glucagon secretion. ​ - We can begin by examining the glucose sensor of the alpha cell. In contrast to the beta cell, this "sensor" is comprised of GLUT1 and glucokinase. This implies that glucose entry into the alpha cell will occur at lower levels than in the beta cell. (Recall that GLUT1 has a Km of about 1mM and that the glucokinase’s Km for glucose is around 5.5 mM). Accordingly, uptake of glucose and initiation of glycolysis will start at lower blood sugar levels. The glucose sensor in the alpha cell is, therefore, responsive to changes in blood glucose concentration in the lower physiological range. Expressed simply: the beta cell glucose sensor responds to increases in blood glucose, the alpha cell's sensor to declining blood glucose levels. Downloaded by Casey Garnett ([email protected]) lOMoARcPSD|36642292 ​ - As you can understand now, secretion of insulin and glucagon are extremely complicated processes. Many elements play a role in determining the sensitivity of alpha and beta cells to plasma signal substances. ​ - Changes in amino acid sequence in one glucose transporter (GLUT1) results in a rare genetic condition where not enough glucose is transported to the brain, especially during development ​ - Glucose is the primary source of carbon and energy for the brain. ​ - GLUT1 deficiency syndrome is a disorder affecting the nervous system that can have a variety of neurological signs and symptoms. o Approximately 90 percent of affected individuals have a form of the disorder often referred to as common GLUT1 deficiency syndrome. o These individuals generally have frequent seizures (epilepsy) beginning in the first months of life. o In newborns, the first sign of the disorder may be involuntary eye movements that are rapid and irregular. o Babies with common GLUT1 deficiency syndrome have a normal head size at birth, but growth of the brain and skull is often slow, which can result in an abnormally small head size (microcephaly ). o People with this form of GLUT1 deficiency syndrome may have developmental delay or intellectual disability. o Most affected individuals also have other neurological problems, such as stiffness caused by abnormal tensing of the muscles (spasticity), difÏculty in coordinating movements (ataxia), and speech difÏculties (dysarthria). o Some experience episodes of confusion, lack of energy (lethargy), headaches, or muscle twitches (myoclonus), particularly during periods without food (fasting). ​ - Diabetes is a chronic, metabolic disease characterized by elevated levels of blood glucose (or blood sugar), which leads over time to serious damage to the heart, blood vessels, eyes, kidneys and nerves. ​ - The most common is type 2 diabetes, usually in adults, which occurs when the body becomes resistant to insulin or doesn't make enough insulin. In the past three decades the prevalence of type 2 diabetes has risen dramatically in countries of all income levels. ​ - Type 1 diabetes, once known as juvenile diabetes or insulin-dependent diabetes, is a chronic condition in which the pancreas produces little or no insulin by itself. For people living with diabetes, access to affordable treatment, including insulin, is critical to their survival. ​ - Diabetes, often referred to by doctors as diabetes mellitus, describes a group of metabolic diseases in which the person has high blood glucose (blood sugar), either because insulin production is inadequate, or because the body's cells do not respond properly to insulin, or both. Downloaded by Casey Garnett ([email protected]) lOMoARcPSD|36642292 ​ - Patients with high blood sugar will typically experience polyuria (frequent urination), they will become increasingly thirsty (polydipsia) and hungry (polyphagia).  ​ - Type 1 diabetes (formerly called juvenile-onset or insulin-dependent diabetes), accounts for 5 to 10 out of 100 people who have diabetes. o In type 1 diabetes, the body's immune system destroys the cells that release insulin, eventually eliminating insulin production from the body. o Without insulin, cells cannot absorb sugar (glucose), which they need to produce energy. - Type 2 diabetes (formerly called adult-onset or non-insulin-dependent diabetes) can develop at any age. o It most commonly becomes apparent during adulthood. But type 2 diabetes in children is rising. o Type 2 diabetes accounts for the vast majority of people who have diabetes-90 to 95 out of 100 people. o In type 2 diabetes, the body isn't able to use insulin the right way. This is called insulin resistance. o As type 2 diabetes gets worse, the pancreas may make less and less insulin. This is called insulin deficiency. ​ - Insulin resistance is when cells in your muscles, fat, and liver don't respond well to insulin and can't easily take up glucose from your blood. As a result, your pancreas makes more insulin to help glucose enter your cells. ​ - Although the risk factors for type 1 and type 2 diabetes are quite different (innate vs. lifestyle) the symptoms are essentially the same. ​ - Kidneys function in renal glucose reabsorption in which filtered glucose is recovered, preventing it from disappearing from the body through the urine. The presence of glucose in the urine is a condition known as glucosuria that is associated with diabetes. ​ - Diabetes is the leading cause of end-stage renal disease (ESRD) in the U.S. ​ - “Life expectancy is known as the number of years a person is expected to live. At age 50, life expectancy is 6 years shorter for people with type 2 diabetes than for people without diabetes. By meeting type 2 diabetes treatment goals, life expectancy can increase by 3 years, or for some, as much as 10 years.” ​ - Life expectancy for type I diabetics: o Porcine and bovine insulin differ from human insulin by just a few amino acids and can produce the same cellularactivation cascades that human insulin does. o The biggest breakthrough came in 1921 when Frederick Banting and Charles Best conducted a series of experiments one summer in the laboratory of J.J. R. Macleod at the University of Toronto. Downloaded by Casey Garnett ([email protected]) o o o Like Minkowski and von Mering, they showed that removing the pancreas from dogs made them diabetic. Then they went a step further and painstakingly took fluid from healthy dogs' Islets of Langerhans, injected it into the diabetic dogs and restored them to normalcy - for as long as they had the extract. With the help of a biochemist colleague named J. B. Collip, they were then able to extract a reasonably pure formula of insulin from the pancreas of cattle from slaughterhouses. lOMoARcPSD|36642292 - There is only a single amino acid difference between human and porcine insulin and only three amino acid difference between humans and cows. o None of the cysteines are altered so the basic shape and charge of the different forms of insulin are pretty much unchanged. o In fact it is only the terminal amino acid (Threonine vs. Alanine) that is a significant difference. ​ - Eli Lilly (July 8, 1838 – June 6, 1898) was an American soldier, pharmacist, chemist, and businessman who founded the Eli Lilly and Company pharmaceutical corporation. o In the 1920s the company established insulin factories next to slaughter houses to extract the precious insulin. ​ - Type 1 diabetes is usually diagnosed in childhood. Many patients are diagnosed when they are older than age 20. Patients may be very lean. In this disease, the body makes little or no insulin. ​ - Daily injections of insulin are needed.  ​ - Type 1 diabetes is a type of autoimmune disease in which the body selectively destroys beta cells of the pancreas. ​ - Leonard Thompson, at the age of 13, was the first person to have received an injection of insulin as a treatment for Type 1 diabetes. He went on to live an additional 13 years, well beyond the expected life expectancy for a diabetic in the 1920s. ​ - Mortality for individuals was found to have declined significantly in patients with Type 1 diabetes in 1965-80 compared to those diagnosed in 1950-64, with 30-year mortality being 11.6% and 35.6% respectively. o A better form of insulin became available. o Monitoring of blood sugar levels has improved. ​ - Bovine insulin differs from human in only three amino acid residues, and porcine insulin by only one. ​ - Insulin is present in all vertebrates and even some versions from fish can be used in human therapy. ​ - Any foreign protein can serve as an antigen and trigger an allergic reaction. Downloaded by Casey Garnett ([email protected]) lOMoARcPSD|36642292 ​ - Patients treated with bovine insulin develop antibodies against this foreign peptide. ​ - Lente is comprised of 70 % long-acting Ultralente insulin and 30% short-acting Semilenteinsulin. o So proportions of a long-acting and short-acting insulin are being combined to produce a intermediate-acting insulin. o Both Ultralente and Semilente insulin are suspended by adding zinc and the size of their respective insulin crystals. o The largest insulin crystals are those of Ultralente, while Semilente contains the smallest, or microcrystals. o Simply put, you're combining a long insulin and a short insulin to make an intermediate-acting one. ​ - The likelihood of immune rejection would be greatly reduced if we could use insulin obtained from gorillas, chimps, or bonobos. But this is not practical.  ​ - Kidneys function in renal glucose reabsorption in which filtered glucose is recovered, preventing it from disappearing from the body through the urine. ​ - The presence of glucose in the urine is a condition known as glucosuria that is associated with diabetes. ​ - Glucosuria leads to excessive water loss into the urine with resultant dehydration, a process called osmotic diuresis. ​ - When blood sugar levels rise well above a target range—which can occur in type 1 and type 2 diabetes—the kidneys often release some of the excess sugar from the blood into the urine. ​ - Using the human gene for human insulin, bacteria or yeast can be transformed to make the human form of insulin. ​ - The ability to manufacture human insulin through recombinant techniques means that immunoreactions to animal insulin is reduced and treatment improved dramatically. ​ - Plasmids used in genetic engineering are called vectors. ​ - Plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to multiply (make many copies of) or express particular genes. ​ - Note, a plasmid vector should not be confused with a pathogen vector such as a mosquito. ​ - While GMO insulin is well tolerated by most people, some diabetics do not react well to it. ​ - The most problematic thing about managing diabetes is the large swings in glucose levels. ​ - Since these must be monitored throughout the day and the proper amount of insulin administered, the body is constantly going from too high to too low rather than staying close to the set-point. ​ - The optimal “set-point” for blood sugar levels is between 80-110 mg/dL. Downloaded by Casey Garnett ([email protected]) lOMoARcPSD|36642292 ​ - The ability to monitor and adjust blood glucose levels quickly is important if one is going to try and keep them near the recommended set point of 80-110 mg/dL. ​ - The huge swings and peaks observed in a diabetic patient is what typically produces the long term health problems associated with the disease. ​ - Why is the monitoring of glucose in the urine not recommended for diabetics as a way of monitoring blood sugar levels? o Urine can be stored in the bladder for many hours.  o Glucose only gets into the urine when blood sugar levels are very high. ​ - A recent study showed that intensive management of blood sugar levels can result in a significantly decreased mortality rate among type I diabetics. ​ - With frequent monitoring and administration of proper amounts of insulin blood sugar levels can be kept within a normal range. ​ - An insulin pump can deliver an appropriate amount of insulin when needed. ​ - Coupled with improved methods of measuring glucose a more normal range can be established. ​ - Continuous Glucose Monitoring - CGM systems usually consist of a glucose sensor, a transmitter, and a small external monitor (which may be built-in to an insulin pump or a stand-alone device) to view your glucose levels. ​ - Blood Glucose Meters (BGM) measure glucose levels at a single moment in time, while Continuous Glucose Monitoring (CGM) systems continually check glucose levels throughout the day and night and can alert you if your glucose levels go too high or too low. ​ - The glucose sensor measures glucose in the fluid surrounding cells. Most of the time glucose travels from your blood vessels into the fluid. Blood glucose (BG) meter readings and sensor glucose (SG) readings, will be close but will rarely match exactly. ​ - Type II diabetics can also be hypoglycemic (low glucose levels) or hyperglycemic (High glucose levels) and so CGM can benefit them as well. -