[MUSIC]. Our vision is to find a cure for type one diabetes, and the inspiration we have to do this is to develop a product that maintains a normal blood glucose level without Insulin treatment, just as we heard in the patients with successful islet transplantation, and we're doing this by using beta cell replacement therapy based on the important protocol that we just heard, but then actually relying on stem cell technology rather than that of organ donor islets. We also wish to combine this with an encapsulation strategy so that you can avoid the use of immuno-suppression, which is required in conventional islet transplantation. This slides illustrate the tip of the iceberg of the type one diabetes patients. This is indicating the small fraction of the brittle diabetic, diabetes patient's, that I mentioned previously that are listed on top of this pyramid. They are in the US and the EU estimated to about 16,000 patients in total, and those patients probably today represents the most severe on med, medical need. They are living a life that is very tough. This is the focus of our endeavors, and if we prove successful in those patients, it could likely be applied or utilized more broadly in type one diabetes progressing first to the group also mentioned previously. Of type one diabetes patients with hypoglycemic unawareness and eventually in, in general use in type one diabetes patients. So, we are relying on what is called the pure epotent stem cells as our source of material, and you could say that these cells are the gift of nature. A single culture of pluripotent stem cells can multiply indefinitely and has, at the same time, the ability to differentiate and become any of our specialized cells in the body. The vast majority of stem cells in our body are already pre-specified to cover particular repertoires of differentiation. They are called the multipotent stem cells, or tissue stem cells, or adult stem cells. They usually reside in a niche where they can actually be dormant for a long time or they can receive a signal, that they should undergo mitosis or cell division and they are able to undergo what is called an asymmetrical cell division so that one daughter cell, actually will remain in the niche. To replace the mother cell while the other cell will bud off and become a, a new cell that can leave and actually be destined for differentiation. This is very well know from our bone marrow stem cells where the residing stem cells in the niches, they reside in the bone marrow, but upon cell division, one cell will maintain the stem cell population while the other can become a circulating blood cell of any of our types of blood cells. So the stem cell is thus maintaining its present within the niche throughout the life, and it's sort of a way to secure tissue replacement throughout, throughout life. In certain diseases and certain pro, processes where you have destruction of cells, that may not normally be replaced. This could actually be due to deficiency in the normal regenerative system, and we know that beta cells do not come back once they are destroyed by the immune system. If this is part of the explanation for the disease of diabetes, we don't know, but so far, it has been hard to actually generate beta cells from in vivo stem cells. Newer data has shown that beta cells may be transdifferentiated from other tissues, but this is the topic of another talk. The pluripotent stem cell can also be called the mother of all stem cells, since it can be differentiated into any of the multipotent stem cells and then further into mature cells. Human embryonic stem cells are known as the golden standard for pluripotent stem cells, also abbreviated HESC. And it was recently demonstrated by the Nobel Prize winner, Professor Yamanaka from Japan, that any mature cell type can actually be reprogrammed using just a handful of factors back into a state of pluripotency that is very similar to that of human embryonic stem cells. This is now known as induced pluripotent stem cells. Both of them are pluripotent. The embryonic stem cell is derived from the inner cell mass of the human blastocyst, and super numerous human blastocysts are usually the result following, following in vitro fertilization, services that are provided to patients that have reproductive problems. Such excess blastocytes by legislation in most countries eventually destined to destruction. For instance, in Denmark they can be cryo-preserved in cell banks for up to five years, but they may be donated, by for generation of embryonic stem cell line. And this needs, a informed consent, by the parents. So the donation is based on informed consent by parents. So parents thus have the potential, if they wish, to actually donate these supernumerous blastocysts for purposes of research. And also thereby for purposes of allowing the opportunity, to develop potential future cell therapy based on derived pluripotent stem cells. Fortunately, such donations, have, have, never been a limitation for the purposes, and remember the supernumerous embryos would have to be destroyed anyway if they are not donated for research. So embryonic stem cells is the source we have chosen as stem cells that we embark on for our project. We are also using iPS, but they are not ready for therapy yet. And we are currently based on developing cell therapy for treatment of diabetes, type one diabetes, based on embryonic stem cells. The aims we have are three-fold. First, we want to generate fully functional beta cells from human embryonic stem cells to facilitate the development of cell replacement therapy as a viable treatment of diabetes mellitus. And, as I just mentioned, we are targeting initially the small population of brittle diabetes patients that are in the most severe need of a therapy like this. The second goal is to evaluate and select encapsulation technology, which is currently considered crucial for the implementation of cell replacement therapy. This will allow us to implant the cells, but also to replace new cells if necessary. And also to actually terminate a transplantation as we can remove the device or the encapsulation if it should be important. Most importantly, the encapsulation will eliminate the need for life long immunosuppression for such patients as the device should protect both against the auto and allo immune reactions. While still allowing nutrients and oxygen and insulin to freely enter and exit the protecting membranes. The circle is to establish what is called GMP Good Manufacturing, eh, practice a compliant human embryonic stem cell line. That can be used for the final production and manufacturing as such, and this is, in itself, a gigantic task that is not as simple as it may sound. A fully compliant GMP cell line is a prerequisite for the final approval for a cell therapy product by national and federal drug administrations. This diagram illustrates the project overview. The big challenge is the directed differentiation of beta cells followed by encapsulation. Understanding control of the biology of human development is key to be able to translate and control similar processes in tissue culture dishes in vitro. Fortunately evolution tells us that in terms of organ development, all vertebrates are very similar, and particularly mammalian species such as mouse and men are very similar in the processes of developing our organs. We have thus learned an awful lot, studying mouse development, and,in particular, mouse pancreas development. As a project overview, we have here a diagram of human embryonic development, from left to right. It's the upper panel, with focus on the cells that eventually become beta cells. And residing in the islets of langerhans. The lower panel briefly illustrates the principle of direct differentiation, where the starting material is the pluripotent stem cells. With all of its multitude of potentials to become anything, they are subjected to sequential treatments to gradually direct them to become mature and fully functional beta cells. In other words, the goal is to replicate, or mimic, the normal developmental process in a body into a dish, developing human embryonic stem cells into beta cells in vitro. The mature therapeutic beta cells would then be encapsulated before the implantation. It's a little more detail in the next slide, where some figures from developmental biology has been included. The inner cell mass from this supernumerous blastocyst is used to generate a human embryonic stem cell culture. The uniqueness of such a culture is that it can actually be cultivated indefinitely. It can multiply indefinitely, so it means that a single culture can be expanded to essentially metric tons. So you could say that a single cell, isolated from a single super numerous blastocyst could potentially serve the entire human population with cell based therapy, not only for diabetes, but for any other given disease that could be treated by cell replacement therapy. In contrast to having to rely on organ donors derive, or, or, organ donor derived cells or tissues, then embryonic stem cells fulfill the criteria of providing an unlimited resource and source from which viable cell types and tissues can be derived continuously. The top panel shows the blastocyst that first undergoes the so-called gastrulation process. That actually is the first major process in all vertebrae developmental biology, where the inner cell mass organizes itself into what is called three germ layers, namely the definitive endoderm, the mesoderm, and the ectoderm. For simplicity, only the definitive endoderm is depicted in this mid-panel of the cartoon, and the tissue ultimately gives rise to the pancreas. In general terms, the endoderm is our inner surface, it means that it is our intestinal tract. Uhh, it includes our stomach, our small and large intestines, as well as the organs that bud out from this gut tube, as we call it. And this includes our lungs, our liver, and our pancreas. The ectoderm, gives rise to all of our outer surfaces, including our skin, but it also gives rise to our nervous system, and the brain, and the mesoderm gives rise to the best of the tissues between the ecto- and the endoderm. And this includes our bone, our cartilage, our muscles, heart, kidney, blood vessels, etc. The cup shapes form. The blue cup is our early, very early, definitive endoderm after castration, at this state, and what actually then happens here is that if you take your fingers and wind out this cup, and pull them apart, you sort of get the next structure that illustrates the early formation of the very primitive gut-tube. And in both ends, they will actually grow together to eventually fuse and become a real tube. Eventually, a mouth opening will develop in one end and an anus will, opening will develop in the posterior end, and we will have the complete digestive tract, where our future food will pass through. All of this is extremely well-described in developmental biology in vertebrates. Also, to include the formation of the endoderm derived organs. On two particular sides in the region of this early gut tube, there will becoming two butts that actually decides to become the pancreas. On the dorsal part, the upper part of the, the gut tube illustrates, the dorsal part of the pancreas initiating and the green part below of the butt, like half of the butt is indicating the other part of the pancreas. And this part also actually the part that is not staying green actually develops into the entire liver and our gall bladder. At the end, we have two small pancreatic "anlagen", as they were called, that come out, and they will eventually rotate and fuse together to make one organ leaving only one connection to the intestine, now the duodenum. And this is knows at the common pancreatic bile duct where also the bile acid will empty into the intestine. And the yellow part, in the next sequential figure exemplifies the later exocrine part, which becomes the major part of the pancreas, where all the digestive enzymes are produced and secreted into the duct and entering into the intestine. In the deudeum, where the food will be processed and digested by all the enzymes produced by the pancreas. The red dots in the yellow pancreas indicates the formation of the islets of langerhans, including the beta cells. And those, of course, are interesting in endpoint cells. So, essentially, we are not interested in any of the other cells but the red dot cells. Islets of langerhans is shown in the upper panel where the beta cells are stained in red and the glucagon cells are stained in green. The lower panel, as before, represent the major channels namely attempts to define these conditions that will allows us to sequentially reproduce similar steps of differentiation. The goal, of course, is eventually to end up with the beta cells and the first step of format of forming the definitive endoderm has been solved effectively by many groups around the world. The factors include active and wince that are critical here and I'll not go more into this, but I would like to approach the next step of the early pancreas into the information, where we have taken use of unique expression profiles of particular transcription factors, that are essential for the pancreas and the beta cell formation. Two such factors are PDX1 and NXX6-1. During the fetal stages of vertebrae development, these two factors are only, and I repeat only, co-expressed in the cells that holds the potential to become hormone-producing islet cells. Moreover, in the fully developed newborn and adult body, the co-expression of these two factors actually only happens in the mature beta cell. The use of flourescent colors, a technique known as indirect imunoflourescence, we can now visualize the expression of these two factors during early pancreas development. With PDX1 in green, and NKX1 in red. When staining the early embryo, the pancreatic buds appear yellow, and this due to the unique overlap of the two colors exactly in this meeting. So only this region is able to co-express the two factors, and you can therefore be sure that if you can, in essence, derive yellow cells in your dish, in your tissue culture dish. If you can find a way to get co-expression of these, then you know that you actually are in a situation that represents the pancreatic progenitor cell. The approach is illustrated in the next slide. And this is actually, you could say project that has been carried out in collaborations also with scientists from Lund University, University as well as a biotech company called Selexus. We are taking advantage of using large scale screens based on dual and multiple visualization of the co-expression of factors, and we are currently optimizing the different sequential steps of the protocol of the direct differentiation. For instance, the two colors for PDX and NKX6.1 have been instrumental in designing our screens in order to optimize this particular step. So if you have a high frequency of pdx cells, we know that we are in the right area of the gut tube namely in the duodenum. We know that these cells potentially can hold the ability to form pancreas, but we have to be able to detect equally high expression of NKX6.1 in order to insure that these cells will represent the pancreatic progenitor cells and even the pancreatic endocrine progenitor cell. In this situation, only a slight fraction of the green cells also positive for the red color, NKX6.1. You can see that many fewer cells express NKX6.1 compared to PDX1. The following slide show a result of such a screening where we have a situation with an optimal hit, as shown in the upper corner. Where more than 40 percent of the green cells are now also red. So if this was viewed in a dual color system, it would appear yellow. When viewed in the red filter setting we can see all the red and can actually find one cell's very similar to the PDX [INAUDIBLE] cells. This is a low resolution picture, but the very similar staining patterns indicating, indicates the presence of a high frequency of pancreatic endocrine progenitive cells. So this hit represents a great progress compared to the benchmark protocol that is listed in the lower panel. And now using similar screening strategies, we have in the subsequent steps been able to identify hits for introducing high level and high frequencies of the cells that are now able to become endocrine, meaning that they will become hormone producing cells. And they will choose the islets phenotype or the islets of langerhans phenotype, rather than becoming endocrine inside purchasing cells. This is implicated by the transient co-expression of Neurogenin3. This is a new marker and a permanent turn-on of NKX2.2. Another transcription factor. This indicates that we are in the correct direction. Neuro gene entry is only there for a while, but once it has marked a cell it will become endocrine and differentiate to what's hormone production. This is close, to the final slide, where we have seen, where we will now see a successful differentiation, where we have all of the red cells actually now beta cells expressing insulin and human C-peptide - this is sustaining for human C-peptide. And there is still many cells that has green nuclei, revealing that they are actively turning on neurogenenin3 three to eventually turn into also hormone producing cells, so this is a very successful hit giving us a large number of the end-state cells. And we have now the task to actually do the maturation to the full extent to actually make these cells also becoming glucose responsive. With the data we have so far, we are very confident that this is within reach to achieve this state of fully functional beta cells. Importantly, we also observe the formation to a lower extent, of some of the other hormone producing cells including the glucagon producing alpha cell, and the somatostatin producing delta cells. So this would be the basis for making islets or aggregates that will then lead us to the final slide. And with this I would like to conclude that our program and progress so far, fueled by our expectations, that we are within reach of being able to provide an adequate reliable and unlimited substitute for organ donor eyelets of [UNKNOWN] for the future use of cell therapy. to, to treat type one diabetes and that when residing in a capsule or a device, where sufficient supply of nutrients and oxygen is available, then the encapsulated cells will allow the intelligent and precise dosing of insulin to it eventually establish new glycemia or cure diabetes. This slide illustrates that our cells will first have to prove themselves in being able to re-establish new glycemia or cure diabetes in a diabetic pick. A vital proof-of-concept study in a large animal before moving towards testing for human application. The encapsulation provides protection against immune rejection and will further allow retrieval of the cells if necessary. I would say this was the end of my lecture. Importantly, I think, if we become successful, I think we will make a major impact in the fight against development of complications for this particular group of patients that suffer so severely from unregulatable type one diabetes. I thank you for your attention [MUSIC]
