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Module 1: Cell source, Isolation, Growth, and Differentiation

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Video 1: Types and Source of Cells
So, until now we have been talking about materials which is one of the three different aspectsof Tissue Engineering right.So, we will now move on to the next aspect which is Cells.So, any tissue engineering application you want cells along with the scaffolds many atimes cells are also used by themselves without a scaffold just for regeneration of tissues.So, it is important for us to know what types of cells are being used, where we can getthem and how we can harvest them and whether we can culture them to get more number.So, because we might not be able to get enough numbers just by isolation from host tissue.So, we might have two culture them.So, it is important for us to understand all these concepts.So, for we will first start with the source of cells.So, cells are basically of three different types we have already discussed this earlier.So, you have autologous cells which are cells from the same individual, allogeneic cellswhich are cells from another individual of the same species and xenogeneic cells fromanother species altogether.So, the there are different sources for these types of cells.So, you could either have differentiated cells of the tissue type or you could use a stemcells and fetal cells or embryonic cells or you could use cells which are of a differenttype in the sense that you might be trying to culture you might be trying to engineerbone, but you would probably have osteoblast which is the type of cell present in bonealong with endothelial cells which is not specific for that bone for the tissue type,but it has its own role in tissue engineering right.So, you could use cells of this kind of types.There are also studies where people have shown that one type of cell can be converted toanother type of cell even if it is a somatic cell means without actually taking them tothe pluripotent state you could just trans differentiate them from one to another typeok.So, there are different things which people have worked on.So, we will try to understand some of the fundamentals and take it from there.So, autologous, allogeneic and xenogeneic have their own advantages and disadvantages.With respect to autologous cells the biggest advantages there is no risk of disease transmissionor immune rejection.Whereas, in case of whereas, the disadvantages there is only limited availability and youwould be injuring the site from where you are harvesting the cells.Allogeneic cells have greater availability and are less expensive; however, there isrisk of immune rejection and disease transmission and you would also end up with the heterologouspopulation when you use cells from another individual.So, xenogeneic cells are cells from other species which means they are the most abundantand the least expensive.So, you can actually get it from organisms which can be grown for harvesting these cells,but there is a much higher risk of disease transmission and immune rejection.So, it is important to balance these advantages and disadvantages.So, people do try to use autologous cells as much as possible.However, it is not readily available in many cases what people do is they try to harvestautologous cells and then culture them expand them to get desired numbers and then use itfor treatment of the injured site.So, in the introduction lectures we had actually talked about using autologous cells for chondrocyte,autologous chondrocytes for regeneration of cartilage right.So, those kinds of things are very commonly done even today.So, differentiated cells and stem cells also have their own advantages and disadvantages.Differentiated cells have the required functionalities which are needed for the tissue to functionthe way we expect them to.Disadvantages you would cause donor site morbidity and in vitro growth is actually more difficult,it does not, you cannot culture them for multiple passages they actually die after some timeor they are more expensive.Stem cells on the other hand are less expensive and there are different sources from whichyou can get stem cells, they can be used for many applications.If you have one type of stem cell then you can try to differentiate it into differentcells and use it for different applications.So, those are some of the advantages of stem cells; however, the disadvantages they donot have the functionality, which means you need to differentiate them properly to getthe desired functionality and they may not do that they may not differentiate as required.So, there could always be an issue with differentiation especially when you have any expect differentiationto happen in vivo.So, in vitro you have a lot more control with what the cells are exposed to and how youactually treat the cells to get the required differentiation.whereas, in vivo the level of control you have is lesser ok.There is also a risk of uncontrolled growth and differentiation.This is especially true for embryonic stem cells which might lead to formation of teratomaswhich could be a problem.So, the limited availability of differentiated autologous cells is one of the limitationswhy people have started people looked for stem cells and other sources and morbidityand limited proliferative capacity are serious limitations when you are talking about expandingthe cells, as I was saying we would not be able to harvest enough cells right.So, if you are trying to harvest a lot of cells then you will cause significant damageto the donor tissue.So, you try to harvest only smaller numbers and then you have to expand them and expandingthem is also a problem because they do not grow as rapidly and they only multiply fora few passages they will not multiply for a long time; so, the that is a problem.So, because of that using autologous differentiated cells is not very easy for many applications.So, with respect to undifferentiated cells the limitations are different for the differentkind of stem cells you use.With embryonic stem cells there is a chance of teratoma formation, teratoma is a tumorwhich tumor containing tissues which is derived from three embryonic layers which is the endoderm,mesoderm and ectoderm and there is also a chance of uncontrolled differentiation invivo.And this these are technical difficulties and you also have ethical questions when youare talking about embryonic stem cells right.Mesenchymal stem cells have a problem because they failed to differentiate into desiredcell types in vivo even though it people have repeatedly shown that in vitro differentiationis very successful.So, because of this it is not very easy to use these stem cells for in vivo applications.So, many a times what people do is they use stem cells and differentiate them, so theyexpand them and then differentiate them in vitro and finally, then place them in thesite of implantation.So, those are some of the challenges with stem cells.So, what are stem cells?So, stem cells are cells that have the ability to divide for indefinite periods of time inculture and can give rise to specialized cells.So, they themselves do not have any special functions.So, they are undifferentiated and they can differentiate into different types of cells.So, there are three different types of stem cells they are called as a totipotent, pluripotentand multipotent stem cells.So, totipotent stem cells are the cells which can form all types of cells in the body andthe extraembryonic cells.So, which are the placenta right.So, it can form all of that and embryo and the first few divisions of the embryo arethe totipotent stem cells which can actually form any type of tissue.So, the totipotent stem cells have the ability to actually develop into a whole organismwhereas, the pluripotent stem cells can form all the cell types in the body, but they cannotactually form the extraembryonic or the placental cells.So, those are the pluripotent cells stem cells.So, embryonic stem cells and induced pluripotent stem cells are examples for such pluripotentstem cells.Multipotent stem cells are the ones which can develop to more than one cell type, butare more limited than pluripotent stem cells they cannot form all the cell types, but theycan form a significant number of cell types.Sir at stem cells in umbilical called multipotent.Yes.So, the cord blood cells are multipotent stem cells, only embryonic stem cells are pluripotent.So, totipotent cells can differentiate into extraembryonic membranes and tissues and theembryo and all the post embryonic tissue and organs which means it basically has all thepotential to develop into a whole organism.So, embryo is basically a totipotent cell.So, the stem cells are broadly classified as adult stem cells and embryonic stem cells.Based on where you get it from right.So, if you are getting it from an adult organism then it is an adult stem cell and if you aredeveloping an embryo from which you are harvesting these stem cells, then it is called an embryonicstem cell.Adult stem cells are actually present in different parts of the body.So, they are multipotent and these were discovered almost 50 years ago and more than 50 yearsago now.Embryonic stem cells are found in developing embryos and fetuses, these are pluripotentand these were first isolated in 1998, they can multiply more readily and seem far moreproficient compared to adult stem cells.So, when you are talking about the three major types of stem cells the totipotent, pluripotentand multipotent.So, the potency of the totipotent stem cells is much higher than that of pluripotent andmultipotent and cell types which are capable which they are capable of generating totipotentcan generate any type of cell including the extraembryonic cells whereas, pluripotentcan differentiate to form all the three germ layers which we talked about.So, whereas, it cannot form the extraembryonic tissue and multipotent is limited to certaincell types.So, examples are zygote and early morula which is the first few cell divisions of the embryoand embryonic stem cells would be pluripotent stem cells and induced pluripotent stem cellsare also pluripotent.Hematopoietic cells, mesenchymal stem cells, neural stem cells are all examples of multipotentstem cells.So, these are basically different types of adult stem cells and totipotent stem cellsare found in the fertilized egg and the early cells of the fertilized egg and pluripotentstem cells which is the embryonic stem cell is found in the inner mass of a blastocystand embryo develops for a few days and forms something called a blastocyst which is a cellmass.The inner cell mass of this blastocyst is embryonic stem cell and multipotent stem cellsare obtained from different tissues.The advantage of each of them as totipotent stem cells are easy to isolate and grow andpluripotent are also easy to isolate and grow.So, you can get a lot of them you can easily be cultured, but with multipotent cells thereis less ethical issues and there is less chance of immune rejection because there is a potentialfor it to be taken from the same person.So, you can get an autologous stem cell and actually culture it and use it.So, cord blood cells and things like that that is why people store cord blood hopingthat they would be able to use these multipotent stem cells eventually.So, the disadvantages with totipotent and pluripotent is the ethical issue and withpluripotent stem cells there is also a challenge of teratoma formation and multipotent stemcells are very difficult to isolate and they have limited differentiation and they arenot available in large numbers.So, when you aspirate blood from bone marrow, It is not very easy to get stem cells youget only a very few cells stem cells for the aspiration you get.So, those are some of the problems.So, teratoma is like a cancer tissue which contains tissues from all the three derm layers.So, it can it is a problem with embryonic stem cells specifically and IPSCs may alsohave the same problem, but people are trying to understand that.So, see adult stem cells are primarily obtained large sources the umbilical cord blood ortissue and there are also cells from brain, cornea, retina, heart, fat, a skin, dentalpulp, bone marrow, blood vessels, skeletal muscles, intestines everywhere right.So, these are just places where you get different adult stem cells and the number of cells youharvest will depend on the source itself right.So, umbilical cord; obviously, has the most abundant and bone marrow has significant numberswhereas, if you were to take it from dental pulp or for other places the numbers are usuallysmaller.Sir I am not sure I have read somewhere that recently the isolated stems cells from teeth.That is the dental pulp.Oh.So, the teeth has a pulp in the route and that is where you can actually harvest itfrom.So, embryonic stem cells are obtained from fertilized egg usually through in vitro fertilization.The ovum has had nucleus removed and the nuclear membrane material is injected from the intendedrecipient which is called it can be reproductive or therapeutic cloning to get the in vitrofertilized eggs which you culture for a few days and then finally get it.So, the question is always about the ethics of this.So, some of the unique properties of stem cells is they are capable of dividing andrenewing themselves for long periods.So, stem cells can multiply for many months in the lab and they are capable of long-termself renewal in the sense that even when they are differentiating, they will maintain thenumbers and they are un specialized which means they do not have any specific, tissuespecific structures.So, they can actually be differentiated into different types of cells.They can give rise to specialized cell types which are which will happen through the processof differentiation.So, differentiation actually does not happen as one step, it goes through multiple stepswhere the cells become more and more specialized and finally, they get committed to one lineageand final form or one particular type of cells.So, adult stem cells are typically generate the cell type of the tissue where they areobtained from.So, if you take hematopoietic stem cells it will form different types of blood, bloodcells right.So, it can be differentiated into other cell types by forceful differentiation, but ingeneral its it can differentiate only to those types of cells.

Video 2: Adult and Embryonic Stem Cells
Adult stem cells are undifferentiated cells which reside in the differentiated tissues.Progenitors and precursors are formed before you fully formed; formed the fully differentiatedcells these are the intermediary steps of the differentiation process.These are progenitors are; however, committed to a lineage whereas, the stem cells are not;so that is the difference.So, although in some cases people do try to use them interchangeably it is not scientificallycorrect progenitor is already committed.So, one example would be endothelial progenitor cells.So, these can only form endothelial cells right, but they are not fully developed intoendothelial cells, but they can actually grow at a much faster rate compared to endothelialcells.So, the function of adult stem cell is to maintain tissue homeostasis and replace thecells which are lost or due to normal tissue turnover injury or disease ok.Bone marrow is where the first set of stem cells were identified and hematopoietic stemcells were identified in 1961 and 63 and these can form the RBC, WBCs and platelets.And bone marrow stromal cells were identified in 1970 and they have been shown to form bonecartilage fat and hematopoietic supporting stroma and so on.So, stem cells have the self renewal property because of something called asymmetric division.So, what happens is whenever you have asymmetric division there are two daughter cells whichcan have two different cellular fate, when you have mitosis the two daughter cells formedare exactly identical and they have the same fate even with me is that is what happensright meiosis also you form two daughter cells which have the same fate they can only begametes they cannot be anything else.Whereas, here that is not what happens, you have asymmetric division where the stem cellswhich divide one will be a stem cell which means the self renewing property is maintained.So, one stem cell forms one stem cell and another cell which can get differentiatedinto any type of cell.So, your cell stem cell population remains the same ok, the number of stem cells in theirpopulation remains the same.So, basically one daughter cell has the same potency which is the self-renewing property,so it is a stem cell and the other one can either be a stem cell or be stimulated forfurther differentiation.So, usually what happens is this daughter cell which has a different fate can actuallymultiply before it gets differentiated that is what happens because after complete differentiationif the multiplication has to happen then it will be much slower.So, multiplication usually happens before complete differentiation.So, that is why these progenitor cells are important.So, these progenitor cells will actually divide at a faster rate, but they are already committedto the lineage.So, they cannot they would not come back to the previous step they will actually get differentiatedto the final type of cells.So, hematopoietic stem cells are the ones which are obtained from the bone marrow.So, the way you harvest them is an invasive procedure; obviously, you have to drill intothe bone marrow to get it.So, it is not a very preferred mechanism for harvesting, but that is the only way you canharvest hematopoietic stem cells.They can also be released into peripheral blood by a process called mobilization.So, this mobilization can actually be induced by treatment with cytokines and thereby youcan actually get them into the peripheral blood and then try to draw them.So, blood from the placenta or umbilical cord would also have hematopoietic stem cells.These are much easier sources to harvest these cells, stem cells.(Refer Slide Time: 20Mesenchymal stem cells are the ones which are extensively studied, they are obtainedfrom the bone marrow of the iliac crest or the femoral head from a patient who is usuallyundergoing a total hip replacement.So, you do not harvest mesenchymal stem cells only for that purpose because it will be aquite painful procedure.So, iliac crust is from your hip and you can get it from your sternum which is the jointbetween your rib cage and you can also get it from the femoral head, femor is the bonein your thigh and the head which gets into the ball and socket joint that is also thatis the head from where you can get these crest.And adipose derived stem cells can be obtained from fat tissues usually which is taken afterliposuction and adipose derived stem cells have also been differentiated to various typesof cells and they have been effective in using for tissue engineering applications.Epithelial cells are usually found in the bulge containing region of the hair follicleand these as I said the numbers would be small it is difficult to harvest them, but theydo have the same multipotency multipotency as mesenchymal stem cells.Other stem cells are neuronal stem cells, multipotent adult progenitor cells and cellswhich are present in your connective tissues and muscle-derived stem cells.So, these are all the different adult stem cells which have been used for different tissueengineering applications.So, multilineage potential of the adult human mesenchymal stem cells was demonstrated in1999 by Pittenger et al and this was published in science and this is one of the extensivelycited papers which has almost 23,000 citations currently and so, this these were these arecells which were previously called as a plastic adherent colony forming units’ fibroblasts.So, the term mesenchymal was coined in this 1999 paper.So, what happened was Pittenger et al demonstrated that these human mesenchymal stem cells havea multilineage potential and they form adipocytes, chondrocytes and osteoblast.So, they also showed that these mesenchymal stem cells had heterogeneity with growth rate,phenotypic plasticity, and colony morphology when cultured in different conditions.So, initially they were identical right.So, when you harvest these mesenchymal stem cells, they are all identical all the morphologyall the properties are the same, but when you culture them in different conditions,they became different cells completely different and that was shown to actually that was shownfor the first time in this paper.So, stem cells are present in your body in a place called stem cell niche.So, this is microenvironment which is well controlled to maintain the stemness of thecells ok.So, this is a specific location in which the cells can reside for an indefinite periodof time without actually differentiating.So, they will they can produce progeny while self renewing in the sense that they can gothrough asymmetric division and the cell which is fated towards differentiation will leavethe stem cell niche and the cell which is going to remain with the same multipotencywill remain in the niche itself.So, this is one of the issues when we handle stem cells.We do not have the stem cell niche outside the body.So, you can have the stem cells, but they do not behave the same way, when you haveit in a stem cell niche, they have certain properties which are, which will be lost whenyou take it out of them.So, there are different types of stem cell niches, so you have a simple stem cell niche.So, these are basically niches where the stem cells are locked to the niche by adherentjunctions and are to the extracellular matrix through different junctions like integrations.So, the niche position of the stem cells is to receive intracellular signals that controlthe growth and inhibit differentiation.So, in a complex niche different stem cells might be localized in the same niche and morecell types can contribute to the niche instead of just one single cell type contributingto the niche.The storage niche is the stem cell niche where the cells are just stored the cells are quiescentinside this these are act as the reserve of stem cells and they are activated only atduring injury or some kind of damage to the tissues.So, that these cells can actually go and repair the injured tissue.So, progenitor cells are cells which are more specific than the stem cells and they candifferentiate for a limited number of times these are not self-renewing, but in some literature,you would see that they are using it interchangeably which is not correct.Endothelial projector cells or pancreatic progenitor cells are cells, which are committedtowards the particular lineage.So, embryonic stem cells were first isolated from mice in 1981.So, it was done by two different groups independently and in 1998 human embryonic stem cells wereisolated by Thomson et al.And these are isolated from the inner cell mass of the blastocyst and can be expandedin the lab while maintaining the pluripotency.So, this is what is done; so, for isolation of embryonic stem cells.So, the what I see to get the embryo out of, so if it is natural fertilization you wouldhave to get the embryo out of the ovarian duct right.So, then that would mean flushing of the duct which will destroy the embryo as well, sothat is not really an option.So, because of this you need to use you need to do in vitro fertilization that is how youcreate these embryos for getting these embryonic stem cells.So, surplus embryos during IVF treatment can be used for this.So, this needs to be donated by the parents after informed consent.So, in India you cannot pay for these things as well.So, whereas, abroad people do pay for some of these donations which people make.So, even when for blood collection in the US, we used to go and give blood very commonlyfor research because many of my friends are working with blood and they would need 30ml blood for researches and they will call one of one of our one of us and we will goand give blood, we will get like 20 dollars or 30 dollars.So, that we used to do that quite regularly, but in India you are not allowed to do thatbecause you can exploit the poor, in the name of doing that.So, in the US you only exploit the grad students.So, zygotes are produced through in vitro fertilization and they are cultured to formthe blastocyst phase and instead of at this stage usually what happens is this is placedinside the womb for it to develop into fetus.So, here instead of that the blastocyst is used for isolation of embryonic stem cells.So, there are only about 200 to 300, 200 to 250 cells in the blastocyst and 30 to 35 ofthem 34 of them as the inner cell mass and the outer layer is removed and you eitherthrough mechanical surgery or through immuno surgeries and after that you take the innercell mass and culture it on a feeder layer and the colonies are mechanically dissectedand transferred to new dishes.So, these need to be cared for daily.So, that you make sure that the differentiation does not happen because when you are culturingit in vitro there could be stresses which are caused that could cause differentiation.So, to maintain its stemness you need to make sure it is cared for and it needs it is maintainedin the way it can be.So, you would always prefer to use this for any still.Sir what was that feeder layer?So, feeder layer is basically another cell, another monolayer of cells which is used onwhich the cells are cultured so.What is that?So, usually they are fibroblasts; fibroblasts are cultured as feeder layers.So, this goes for any cell type.Fresh non frozen cells are always preferred when you are talking about primary cells comparedto frozen and thawed cells.So, the cell lines which you use are different those are immortalized.So, those you can put it in liquid nitrogen and then take it out and use it, but for primarycells you would not want to do that, so that is why you need to harvest them regularly.So, this means it is a problem right it is you are not going to get enough cells withrespect to embryonic stem cells.So, there are also issues with respect to long term stability and there are cases wherethere is early differentiation.So, when you culture these cells in 2D environment there is the cells can actually pile up right.So, instead of forming a monolayer once there is more cells it can start piling up and thiswill create stresses causing differentiation around the edges or in the piled-up areas.So, these places would experience more stresses and it results in differentiation of cells.So, you can actually use endogenous transcription factors to activate the cells differentiationand this using different growth factors using different cytokines and other markers youcan actually control how the cells are differentiated and you can also co culture with cell typeswhich are capable of lineage induction.So, there are studies where people have shown that culturing endothelial sorry stem cellsalong with pancreatic cells, islet cells would cost them to differentiate to form islet likecells.So, those kinds of things are possible.So, IPSCs are the last type of stem cells which we will quickly go into.So, these are basically somatic cells which are differentiated into induced pluripotentstem cells.So, this was first shown in 2006 by Takahashi and Yamanaka and what they showed was therewere a set of four genes which can actually be transmitted into the cells to form theminto pluripotent stem cells.So, human IPSCs were then reported a year later.So, these cells have very similar properties as embryonic stem cells.So, Yamanaka won the Nobel Prize in 2012 for physiology and medicine for this discovery.So, mouse IPSCs demonstrate certain important characteristics which are seen in pluripotentstem cells.Basically, expression of stem cell markers ability for formation of teratomas which aretumors containing all three germ layers and the ability to contribute to many differenttissues when injected into the embryo for at early stages of development.So, human IPSCs also have the similar markers and ability to form cells from all three germlayers.So, there is a lot of studies going on in this direction and it is a very recent developmentright.So, less than 12 years old, so it is a very interesting domain.