| 1 | Miltenyi Biotec | Webinar: Stem Cell Reprogramming | 3426 | 48 | 4 | 55.3 | positive | 32:40 | [Music] I will tell you today something about the introduction uh or I will introduce about the foundations of stem pramming and will try to give you insights into the newest and the most efficient methods for this so let me briefly go into um the agenda for today first I like to start with an explanation what stem sets actually are uh then I want to introduce the technique of reprogramming and uh tell you what it is used for uh I will introduce different reprogramming techniques and we'll go further into detail for one technique namely the MRNA reprogramming and the last point for today will be the verification of ipscs so let's start with the first topic what are stem cells so when we talk about a stem cell what do we mean by that term stem cells are defined by two characteristics the first is their ability to self renew that means that they are able to create an identical copy and this way they can be maintained in a culture so they build a constant pool of stem cells for regeneration the second ability is the capacity to differentiate into specialized cell types usually progenitor stages that are more restricted in their developmental potential and will then differentiate into further specific lineages so here I want to describe shortly um the development of an embryo and show where to obtain embal stem cells um so the whole story begins with an oversight which is fertilized by a sperm the next step in the development will be the formation of a morula and then the next step will be the formation on about day 4.5 of the plastoy and this consists of an outer cell layer here um symboled by these yellow cells and also a blue uh symol Inner Cell mass and from this Inner Cell Mass you can can um separate embryonal stem cells so these blue cells are the embryonal stem cells and these embryonal stem cells have or has a special feature that they can differentiate into all the lineages of a body for example they can differentiate into cells of a circulatory system or the nervous system or the immune system and if you follow this whole process of development then this is what is described as differentiation I also want to uh introduce other terms to you which you will face if you uh are going more into detail for stem cell research um there are different features uh of different developmental stages and the first um feature is the toy potency which describes the ability to form all tissues in the body and the CL Center and this is a feature the Mora has for example and this means that all cells inside the Mora are able to not just form the embryo itself but also extra embal tissue if you go further in the development the next uh feature will be the PLU potency and this is the ability of the embal stem cells the ability to form three germ layers like here displayed the next um feature is the multi potency and this um or this is the term for the ability to differentiate into multiple cells or tissue types the so-called progenitor cells and an example for this is um are the hematopoetic stem cells which can form um uh different cell types of blood but nothing else and the last feature in this development is the uni potency which describes um The Limited differentiation potential into just one cell Lage the so-called precursor cells so as you have now learned where to find stem cells in Vivo how can we produce PL poent stem cell or BL poent cells artificially here you see again what I told you before here we have the prepotent stem cells so embryonal stem cells abbreviated by ESC and they um can form these three germ lay uh germ layers of the body the ectoderm which consists cells of like skin and brain the mesoderm um which can form bone and blood for example and the endo which forms pancreatic cells and lung cells in this process from the cell cells to the differentiated cells is called differentiation and for a long time it was thought that these uh is a one-way Road where you could just uh move forward into a more differentiated stage but however especially with direct reprogramming um done in the yamanaka lab I will explain this to you later on in more detail and then it became clear that you can go back from a differentiated cell into and PR poent stem cell stage and this is um forming then or this process forms then the so-called induced port and stem cells here abbreviated by ipscs here I want to introduce a timeline to you about the most important um Publications about reprogramming and I just want to highlight two of them um the first is um that here uh the first evidence that reprogramming of somatic cells is possible was done in 1962 by John giren and he cloned frogs by transferring the nuclei of differentiated intestinal cells into amphibian o sites and and then pretty much later in 2006 the yamanaka lab found out how to mimic this process with transcription factors and maintain these cells in a PL poent stage so ipscs behave like esls for example in s renewal and PR potency but also show a similar morphology also proliferation Gene expression and epigenetics so why are IPS cells now so attractive because they become um for example the ethical challenges of an es cell as you always have to destroy the embo if you if you isolate the inner semas of the plastoy and these IPS cells are especially interesting to the field of regenerative medicine as it's possible to reprogram patient specific um cells for example of a skin biopsy and they get can get then further differentiated um to study genetic diseases and screen for thetic compounds to find drugs because I promise you it's pretty much better to kill the cells uh with non-working drugs than killing the patient the other way around you can also use if you have a known uh disease mutation you can take this patient specific IPS cells to repair this IPS cells then further go on with differentiation then you have your healthy cells and this you can use for Trans uh Plantation into the patient and uh this eliminates the danger of immune rejection so this slides uses the so-called warington landscape to show the difference between cell States as a sort of an activation energy concept so once the same moves down the slope it differentiates and cannot go back in their normal development so here you see the second differentiating cells and it goes a different way and however as shown with the second ball we programming bypasses this and sends a cell to prow potency and note uh the ball is not shown to travel back along the same path it it tooks to differentiate so you would never exactly know where your cell is in this landscape how this reprogramming process is working in detail is not completely unraveled today but here I want to make it a little bit more clear at the top of the picture the whole story begins with the application of the four transcription factors op four so 2 kale of four and simic and this destabilizes the somatic cells in this case here FR plus and then a big case is occurring a lot of cells begin to do things which we don't want them to do other cells uh begin to establish certain expression profile FES and just a few number of cells and in this um this ass symbl by the spotter neck over here um just a few number of cells starts to establish the expression profile we wanted to have a whole Cascade of protein expression starts while the epigenetic landscape of the cells change and if this occures and the transcription factors shown here are expressed the reprogramming is done and the sematic cells transformed into an IPS cell so let's go for the third Point uh which reprogramming techniques do we have so I just briefly introduced to you the first technique which was the first evidence that sematic cells can be re pramming uh reprogrammed which has been published in 19 uh 62 by the G lab and he performed nuclear transfer from differentiated intestinal cells um into amphibian Ides which leads to uh cloning of frogs and they showed that all genes needed to make an organism are present in A specialized cell so this was maybe you know the story uh this was also done um with a cheap Dolly so just the cloning of an animal um the next method is the sell Fusion which is an alternative approach to reprogram the somatic genome which involves the creation of hybrids between somatic cells and other cells that contain reprogramming activities and um the special feature is or um the advantage is that the pro poent phenotype seem to dominate also in somatic sets so if they uh divide over here then always um the prepotent phenotype is um obvious but unfortunately it has been shown that this method is very inefficient and the last method I want to introduce is the direct reprogramming where we have the introduction of a small number of transcription factors of the yamanaka factors and um then the cells are reprogrammed and this is now the gold standard to produce ipscs and we will go further into detail during this presentation so there are two different groups of method methods which exist for direct reprogramming one that includes carrier for the transcription factors that integrates into the genome so here here you see that so later on the genome is altered and one method that is integration free and therefore the genomic Integrity is maintained and if you want to go for clinical trials and therapy we of course should go for the integration free method to conserve the original DNA nevertheless I like to shortly introduce two important integrative methods first is the retr rers um which has been used by shanaka um but this method takes up to six weeks to uh form um IPS cells um partial reprogramming so we have two phases of reprogramming partial reprogramming generates class one IPS cells that Express survival trans genes and enog genous po potency genes and full reprogramming in so-called Class 2 IPS cells silences the vector as the endogenous genes maintain the pr poent stage thus retr Vector silencing serves as a beacon marking the fully reprogrammed poly poent stage uh the disadvantage is that you alter the genome and don't have any time any control about the dose and furthermore you don't know if uh the silence genes are reactivated in a later stage um also the efficiency is very low it's below 0.1% and here we have the second um uh method I want to introduce the piggyback transposon system which is a Mobi genetic element that transposes between vectors and chromosomes via a a kind of a cut and paste mechanism and during transposition the pgb transposes recognizes transpose on specific uh sequences located on both ends of the transposal vector and moves the contents from the original sites and integrates them into specific chromosomal sites but the special feature of the piggy transpose and different from the ret Rivers is that it's also reversible nevertheless it is in genetic modification and again we have no final control about the time and dose of the expression of these um transcription factors for the integration free delivery there are also different methods on the market and here I want to introduce two of them the first is where it is here the sendi virus which acts exclusively in the cytoplasm of cells and cannot enter the nucleus or alter host chromosomes and it is therefore fundamentally free of the risk um associated with um conventional viral vectors Sendai viral vectors also reprogram cells with high uh efficiency the disadvantage of using Sendai virus is um that they have relatively high costs compared to um other methods um the establishment of virus free IPS lines requires up to 10 to 20 passages depending on the cell line um and reprogramming conditions and you must isolate and clonally propagate at least 10 primary primary colonies to establish only a few IPS sell lines per patient sample another integration free reprogramming method is the MRNA delivery and here uh it's very important um that you have the ability to reprogram stem sites using a method that is both non-integrative and nonrival and this is very important for future theerotic application and the advantage of this technique is that there is no risk for insertional mutagenesis that can later cause cancer and for CS that will eventually be used in the clinics this will likely be the dominant technology um also a big Advantage is that uh it's very fast this technology and you have a very very high efficiency about over 1% um for example MI a reprogramming can result in IPS cell colonies within two weeks but one disadvantage of um the MRNA reprogramming is uh the inconvenience posed by the daily mRNA transections required for this Method All right so let's go further into detail for this Mna technique uh waret Al published in 2010 how to generate synthetic highly stable Mna which can be used to reprogram ipscs and in their publication they showed um reprogrammed C colonies within up to se uh 17 days so this is what you see here I also want you to introduce uh our uh reprogramming kit the stemx MRNA reprogramming kit uh and we will see one typical workflow for mRNA reprogramming so we start the whole culture with um the preculture of sematic cells like for example fi blasts um in Repro Brew XF medium and then code plates with CTS and pl the cells on top allow the cells to adjust and on day Zero start with the transactions you will go on with the Trans affections for 12 consecutive days um also adding this b18r which was identified to enable increased Sur viability during uh RNA transection and on day 11 after the 12th transaction we stop with the transactions and allow the ipsc colonies to grow then we come to the point where we change the medium to to IPS Brew medium and um IPS Cy colon is are visible and I want to do that with you uh more or less live and this is what you would see through um the fluorescent microscope on day one these are fiber blasts and they have been um transfected for one or two times on day one and the big advantage in our mRNA reprogramming kit is that we use egfp as a transaction control so if these cells are green florescent like you see over here uh this shows directly how uh how efficient this method is and you see more or less 100% of the cells are transfected so here you see day three um the morphol morphology didn't change uh they grew a little bit the celles but still they're more or less 100% green so efficiency is still very high this you can also observe on day five Cs grew nicely uh and here on day seven you see the first sign that the morphology is changing a little bit so here you have more dense Parts in your culture and on day nine it gets little bit more obvious here there are very dense parts and you see maybe that they are um there's a little bit enhancement of the size of the nuclei which is um um normal for is cells but still the efficiency is very high and then very obvious on day 11 this is what you want to get out at the end of reprogramming this is a nicely formed um ipsc colony to be sure for the plal potency of this colonies you can also stain them with OCT 4 uh which serves or which is a transcriptional marker for p poent stem cells and serves like a fingerprint to identify IPS cells and in this case you see very nicely formed green florescent colonies um to keep the IPS cells further in cure you have to isolate them from the differentiated cells because you always have uh as I showed you before a lot of uh different sh or uh other cell types in your dish and uh also IPS cells also dead cells and you just want to go on with your prepotent cells and um one method would be that you manually pick these cells and isolate them so it's very hard I can tell you to pick this um that uh PR poent cells as you don't really know if you have really uh portent cells or not as you just have your dish and uh they are not fluorescent or something like that because normally for a picture like this you have to fix your cells and then you kill your C so you cannot go on with further passaging um therefore you would pick all cells which have a PR poent morphology you would replay them and passage them further on but um a new technique or a new highlight in our product portfolio is um a Life Cell stain antibody U which you can see over here um and for this you don't have to kill your C and you don't have to fix your cell and you can directly under the fluorescent microscope see what you really are or want to pick um one additional problem is that you maybe assume one colony is monoclonal so just originating from one cell but as you see by this picture it's not every time the case the more efficient option to isolate uh the reprogrammed cells is to separate them and this is one very famous technology for miltin biotech um therefore we have the St 160 microbit kit for the isolation of mRNA ipscs and this is working like um I would explain now so you have your uh PR poent cells which are expressing tra 1 60 and we have um very very small micro beads uh which binds to the tr60 positive cells these micro beads are consisting of iron and uh if you put that sample with your pro poent cells and some other cells inside on top of such a column uh where is a steel ball Matrix inside and you place a column into a magnet then these um microb beats um attached to the cells will bind to the column and everything what it's what is not positive for Tron 60 will run through and if you later on release the column from the magnet um then the um uh the column is not mag magnetic anymore and will not bind uh the magnetic beads anymore and then you can also wash out your then positive sample and hopefully have just your trauma 60 positive cells and this we have done over here and also analyzed with our flow cytometer um what is displayed in this plots is um two markers are two markers at first TR 160 um for plor potency and here the epcam marker uh which also serves as a pre potency marker and here on the um on this part of the blot uh you see um cells which are negative for both markers here you see cells which are positive for both markers and here you see cells which are just positive for the epca marker and here you see cells which are just positive for the tri 160 and what we want to obtain later on are these cells so in the original fraction just after uh the reprogramming you have for example here um just 42% of PR poent stem cells and if you now place all these cells also with the negative cells on the column um you get out a negative fraction first of all so what is washed out and don't bind to the cell and uh here we analyzed um how much per poent C cells are still here in this uh fraction and we saw there's just uh very yeah very small loss of cells of 2% so and if we then um analyze a positive fraction we see that the cells are pretty much enriched uh about a value of 94% so these enriched cells you can then later on use to replay them and then you can start uh with a pretty much higher cell number which will fasten the experiments dramatically all right then we come to the last point of my talk today U the verification of ipscs so there are different functional essays of per potency first is the Embry body essay um and these Embry bodies are generated by esc's on nonadherent plates and this is very important in the absence of safe renal cyto kindes so they differentiate and after several days of culture um this is resulting in ID bodies which can be individually transferred and cultured further under conditions appropriate to Coke's development of various cell types and then stained for uh different cell type marker to obtain if they are really um able to form all the uh cells of the body and which which is the evidence that this cells are still important um the second uh essay is the teratoma essay um and teratomas are generated by injection of cells into nonobese combined imuno deficient mice usually intramuscular or under testicular or kidney capsules and after 10 till 12 12 weeks um or also a little bit longer um you have teratomas and and uh they can be recovered and analyzed uh using histological approaches to examine uh as well if you have all the different tissues uh inside all right that's it for the foundations of reprogramming let me briefly introduce where you can receive more support on getting an expert in your stem experiments so we offer a 4day stem set course in our headquarter in bbak in Germany and also in San Diego uh USA where you can learn theoretically and practically to perform a whole stem cell workflow including reprogramming cultivation and differentiation and um maybe you also want to um attend one of our next webinars and then we invite you very much uh to to come to our website milon biotech uh.com weinar or you can also find more trainings on our website under milon biotech.com trining so finally thank you very much for your attention please let me know whether there are any questions thank you [Music] he [Music] | ↗ |
| 2 | Miltenyi Biotec | Overcoming challenges in scaled manufacturing of iPSC-derived cells [W... | 683 | | | 22.0 | | 45:14 | No transcript | ↗ |