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

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Video 1: Hematopoietic Stem Cells
We had looked at tissue dynamics and while we talked about tissue dynamics, we talkedabout what the cells would usually be doing when they are residing in the cell, residingin the tissue and the first thing we talked about would be the cell division.So, which is cells dividing and multiplying and you actually have cell differentiationas a next step usually like if you have a stem cell which is where the most of the tissuesare going to originate from, and the cells would all come from stem cells, rather thanmatured cells multiplying to cause to create large number of cells.Usually for creating new tissues, stem cells are recruited and you actually have stem cellsmultiplying to form the desired tissue; for that to happen differentiation is a crucialfactor.So, we will talk about cell differentiation.So, differentiation is basically the process of cells specialising in a function.So, stem cells as you all know, do not have any specialized function they can mature toform any type of cell.So, the differentiation is the process which results in the formation of a specific celltype with the desired functionality.So, there can be cell differentiation because of the process called asymmetric division.Asymmetric cell division is basically where you have two non identical cells which areformed, the daughter cells are not the same.So, in regular mitosis you would have two cells which are exactly the same.Here, in asymmetric cell division, one is basically for the self renewal property ofthe stem cell.So, it basically divides and retains its stemness, other goes in to the progenitor cells andthese progenitor cells can then become more and more differentiated to form the finaldifferentiated cell lines, but usually these progenitor cells multiply through just regularcell division to form more number of progenitor cells, which then get differentiated.So, this is because as I already mentioned progenitor cells divide at a much faster ratecompared to fully differentiated cells.So, what you are seeing in the figure is the, first step is the asymmetric cell divisionwhere the cells are dividing, one is forming stem cell which is self renewing, the otherone is a committed progenitor cell.The committed progenitor cell basically then multiplies and amplifies.This is the progenitor division so, that you have more number of cells that can be differentiated.And the final step is the terminal differentiation to form the fully differentiated cells.So, during cell differentiation you would experience something called population asymmetry.Basically, divisions of stem cells population results in a large number of cells and thesecan actually form different types of cells and this provides additional versatility.Cells can proceed down several possible fates depending on the environment, they are in,conditions that are provided to them and so on.So, if half the cells become stem cells, the total population of the stem cells remainsa constant.So, that way you have the self renewing property of the stem cells taken care of.So, what actually happens in a differentiation process is, as a stem cell matures it undergoeschanges in its gene expression that limit the cell types that it can become.So, there are changes inside the cell which make sure that it is beginning to get someof the functionalities of the differentiated cells and it then, because this happens itsstarts moving closer towards the final cell type.So, these changes are actually monitored by the, can be monitored by the presence of proteinson the surface.So, this surface markers and receptors which are expressed can be used in identifying whetherthe cell is remaining as a stem cell or if it is moving closer towards a differentiatedcell.So, with each successive change the cell moves closer to the final differentiated cell.And its potential for becoming a different cell is actually becoming lesser.So, it becomes more and more committed towards one particular cell type as more and morechanges happened to the cell.So, which is logical right?So, it is not a one step process, it is not like the stem cells form a committed progenitorwhich multiplies and then miraculously it just changes to one particular cell type,it does not happen that way.It is a sequential process which takes significant amount of time and as they as the time goeson it gets more and more committed towards particular cell type.So, this is an example of how it could actually happen.So, what you see here is a totipotent cell which is non self-renewing like zygote.So, zygote does not self-renew right and then you have the pluripotent stem cells whichare self-renewing and embryonic stem cell would be an example of the pluripotent stemcells.So, then this forms a broad potential self-renewing cell.So, this these are still self renewing cells, these are multipotent stem cells which comefrom here and then it becomes a limited potential, limited self renewal cell which, until thisprocess what you would see is there is a possibility of the cells going back right.So, it can actually go back a stage because it is not fully committed yet.So, it is it can still go back and here it forms some kind of a progenitor.So, here the example given is for neural cells.So, it forms a neural progenitor and here it is more committed towards one particularlineage.So, from here it then divides where it is even more function specific, where it canbecome a neuronal progenitor or a glial progenitor and here by this point it becomes almost irreversible.So, after this it is now committed to a particular lineage and it cannot actually go back.So, and then finally, it forms a neuron or a glia based on how the conditions are.The cells can probably die at any of these stages, but they cannot go back after it commitsitself to a particular lineage.So, this would be how a general process would look like.So, I will talk about a couple of things which are very popular when we discuss stem cellsand cell differentiation.One process which is very well studied and has been very well established is the hematopoieticstem cell.The process of hematopoiesis; the process of hematopoiesis is basically the processwhere hematopoietic stem cells commit and differentiate to form different blood cellsright.So, that is basically the formation of all the types of blood cells is from a type ofcell called hematopoietic stem cell.So, these actually reside inside the bone marrow and they have the ability to give riseto various types of matured blood cells and tissues.So, these are self renewing cells where the daughter cells can either be hematopoieticstem cell or a myeloid or lymphoid progenitor cell.So, once it commits towards a myeloid or lymphoid progenitor, it will then go through the differentiationpass pathway and to form different types of cells.So, but it goes through asymmetric cell division first so, that the self renewing propertyof the hematopoietic stem cells is taken care of.The progenitor cells can follow any differentiation pathway.So, these myeloid or lymphoid progenitor cells are not going to form one type of cell right.So, myeloid will have its own variety of cells and lymphoid it can have its own variety ofcells that can be formed.So, they can follow any of these pathways leading to production of one or more cells,cell types, but these cell types cannot renew themselves.So, these progenitor cells cannot renew themselves they already committed.So, this is a simplistic representation of what hematopoiesis is.So, what you see here is the multipotential hematopoietic stem cell which has the self-renewingproperty, then differentiating, multiplying to basically form a common myeloid progenitoror a common lymphoid progenitor.So, this is formed through asymmetric division.So, you either have another HSC formed or a common myeloid progenitor or common lymphoidprogenitor which is formed.So, from here from the myeloid progenitor, you can get megakaryocytes which form thrombocytes,erythrocytes, mast cells and myeloblast.Myeloblast can then go to form basophils, neutrophils, eosinophils, monocytes and macrophages.A common lymphoid progenitor cells can actually form the natural killer cells or it couldform small lymphocyte which should then further be differentiated to form T cells and B cellsright.So, this is a very simplistic representation of what a hematopoiesis process is.So, in general blood cells are actually divided into three lineages.You have the red blood cells or the erythrocytes these are the oxygen-carrying cells they havethe haemoglobin.So, they bind to oxygen and they can carry oxygen to the site.So, these are functional cells that are released into the blood.So, once a differentiation happens it will get released into the blood.They are formed by the process called erythropoiesis.So, within hematopoiesis there the pathway which is taken for differentiation of hematopoieticstem cells to form erythrocytes is called erythropoiesis.You also have lymphocytes which have a critical role in immune response and especially inadaptive immune systems.So, these are composed of the T cells, B cells and the NK cells or the natural killer cells.So, these are formed by the process of lymphopoiesis and cells from a myeloid lineage include granulocytes,megakaryocytes and macrophages.So, these are derived from a common myeloid progenitor through myelopoiesis.This is also, these cells are also involved in innate immunity and blood clotting andmany other mechanisms they have a versatile functionality in your body.So, these are the three major lineages for blood cells.So, the process of forming granulocytes is called as granulopoiesis or granulocytopoiesisand this is done, this is done inside the bone marrow except for mast cells, where themast cells are actually produced; the differentiation happens outside the bone marrow which is calledthis extramedullar maturation.So, medulla is the technical term for the bone marrow.So, its extramedullar maturation.So, megakaryocytopoiesis is the hematopoiesis of megakaryocytes.So, this is the general hematopoiesis process.So, if you were to look at in a more detailed step by step fashion this is what it wouldlook like.So, the multipoietic, multipotential hematopoietic stem cells have the self-renewing propertyand they form common myeloid progenitors or common lymphoid progenitors as I had alreadymentioned.And so, the common myeloid progenitor can basically come out of the bone marrow andthen mature into a mast cell, that is the extramedullar maturation to form mast cells.So, inside the bone marrow, this myeloid progenitor can then become megakaryoblast, which thenbecomes promegakaryocyte and a megakaryocyte.So, this megakaryocyte then comes to the blood where it forms thrombocytes or platelets.So, this process is the thrombopoiesis where you end up forming the platelets.So, the formation of megakaryocyte is megakaryocytopoiesis ok.So, the other pathway for erythropoiesis would be, it goes into the pro, to form proerythroblasts,basophilic erytro erythroblast and then polychromatic erythroblast, orthochromatic erythroblastor a normoblast and from where it forms a reticulocyte or a pro polychromatic erythrocyte.So, this enters into the blood stream where in the form of erythrocytes where it getsmatured.So, this pathway is the erythropoiesis.So, as you see these are all coming into the blood where as mast cells enter into the tissue.So, the from the common myeloid progenitor you could also have the myeloblast which canthen differentiate into different granulocytes like basophils, neutrophils, eosinophils andit can also form monocytes which can then differentiate to form dendritic cells andmacrophages.So, as you see there are steps for each of them.So, it all goes through the promyelocyte then to myelocyte, then metamyelocyte, then a bandcell finally, forming either a basophil or a neutrophil or an eosinophil and so on.And so, what you need to notice here is where these differentiations happen right.So, until the last step everything happens inside the bone marrow and the final stepis where it happens in the blood, the final maturation happens in the blood or it getsreleased into the blood ok.So, the common lymphoid progenitor; however, forms these lymphocytes which is which goesthrough either a lymph lymphoblast, prolymphocyte and then forms a small lymphocytes which wouldthen develop into B cells and T cells or it could go into forming natural killer cellswhich are the larger grano granular lymphocytes.And you could also have this common lymphoid progenitor forming dendritic cells, the lymphoiddendritic cells directly.So, as you see there is significant number of process steps which are involved in eachof these process it is not like.So, until unlike the previous thing which we saw so, in the previous one what we sawis, it just goes from here to here right.So, myeloid to erythro erythrocyte is what a simple representation is, but in realityit actually has so, many different steps the cells exist in four or five different levelsof differentiation before it finally forms the fully differentiated cell.So, hematopoiesis can actually be modelled and there are two theories for modelling it;one is the deterministic model and another is a stocha stochastic model.So, determi deterministic model is the classical way of describing hematopoiesis, what we kindof saw is the deterministic model where we say it goes into this, this environment willprovide this particular condition where it goes into everything it goes into particularpathway and there is no real randomness to this process.It just depends on the factors, the colony stimulating factors and other factors presentin the microenvironment.So, this determines the path for the cell differentiation right.So, this is what we kind of have always believed in and this is a classical pathway.However recent experiments are supporting the theory of stochastic differentiation.So, basically undifferentiated blood cells can differentiate into specific cell typesby randomness.So, there is no real control over how this happens and there are factors which do control,but there have been studies which show that the cells can go back and at each step andthere is quite a few; there is a significant level of randomness which cannot be completelyignored, you cannot just say that it is deterministic saying this environment will definitely leadto this, it is not that.So, and so, this actually is a representation of the growth factors and other moleculeswhich can guide into specific differentiations.So, what you would see here is many of these factors are repetitive, which is probablyone of the reasons for the stochastic model of differentiation right.It is not that like, for example, this IL-3 can be present in it is present in the formationof myeloid progenitor, which can then form megakaryocytes or erythrocytes.So, it is present in both.So, it could play a role in both these conditions right.So, based on this it is actually very difficult to say that one particular environment isgoing to force cells into dividing into forming particular things.But people can, people are trying to use this understanding to design the cocktail in whichthe cells can be grown so, that there can be differentiation that is directed towardsper specific cell lineage.

Video 2: Mesenchymal Stem Cells
So, the next major type of stem cells which are extensively studied and extensively usedin tissue engineering applications is the mesenchymal stem cells.So, these are basically multipotent stromal cells that can differentiate into varietyof cell types.So, basically they have been shown to form osteoblast, chondrocytes, myocytes and adipocytes.So, osteoblast are bone cells, chondrocytes are cartilage cells, myocytes are muscle cellsand adipocytes are fat cells.So, mesenchymal stem cells have a very good capacity of self-renewing while maintainingtheir multipotency.The capacity of the cells to proliferate and differentiate is known to decrease with theage of the donor as well as the time in culture.So, as even if it maintains its stemness as you have multiple passages, then you wouldsee that the rate of proliferation would be lesser and its ability to differentiate intodifferent cell type comes down.So, the original source for mesenchymal stem cells is bone marrow.So, haematopoietic stem cells are also from bone marrow right; however, these are differentfrom the mesenchymal stem cells, the HSCs and MSCs are different although they bothreside in the bone marrow.Bone marrow mesenchymal stem cells do not contribute in the formation of blood cells;where as hematopoietic stem cells are fully committed towards a formation of blood cellsin your body.So, because they are not involved in the formation of blood cells they do not express the HSCmarker CD34.So, that is actually used to identify whether the cells you have isolated is a mesenchymalstem cell or a hematopoietic stem cell.So, these are also referred to as bone marrow stromal stem cells and so these terms areused interchangeably in many cases.Another source for getting mesenchymal stem cells is the umbilical cord tissue.So, this is the youngest and the most primitive mesenchymal stem cells that is why there isa lot of interest in storing the cord blood and the cord placenta to hope that you wouldbe able to use it for some regenerative medicine which might be developed at in the future.So, this basically has two different tissues one is the Wharton’s jelly and the otheris the cord blood.So, the Wharton’s jelly is a gelatinous material which is present in your placenta,which is, which is very rich in mesenchymal stem cells and the cord blood is rich in hematopoieticstem cells.So, there are other sources as well.Adipose tissue is a rich source of adipose derived mesenchymal stem cells, molar cellswhich are basically the teeth tissue.So, the if you have a developing tooth bud then those are rich in mesenchymal stem cells.So, these mesenchymal stem cells are the dental stem cells actually form enamel, dentin, bloodvessels, dental pulp and various nerve tissues.So, people have also shown that these can form hepatocytes and many other types of cells.Amniotic fluid is also a source from where you can get mesenchymal stem cells.So, it is the fluid in which the fetus is present and this can actually be harvestedusing a process called amniocentesis.Amniocentesis is nothing but taking a large gauge needle and injecting it into the uterusto draw the amniotic fluid.So, this is usually done for diagnostic purposes.So, this amniotic fluid basically has a lot of cells and one in 100 cells which are collectedusing amniocentesis is a mesenchymal stem cells which could also be used for tissueengineering applications.So, this is a representation of how mesenchymal stem cells can actually differentiate to forma various lineage cells.So, I am not going to go into great details.So, what I have here is for adipocyte, myocytes, chondrocytes and osteoblast.So, within that we have basically classifications we have the white adipocytes, brown adipocytes,skeletal muscle and cardiac muscle, smooth muscle and chondrocytes and mature osteoblasts.So, this kind of gives what is the pathways, different pathways these mesenchymal stemcells can take and what would be the growth factors and other molecules which can be helpfulin converting in directing the mesenchymal stem cells into these lineages.So, whatever we looked at right.So, the basic concept of differentiation is what usually happens in your body.So, you want to try to use this to direct differentiation; ultimately that is the aimright.We want to, because we are looking at a tissue engineering application, where we are goingto use these stem cells and we would want to differentiate these stem cells to producespecific cells with desirable functionality.So, that we will be able to perform the tissue we develop will be able to perform its functions.So, this is basically done through activating endogenous transcription factors or it canalso be done by transfection of, it can also be done by transfection of stem cells withubiquitously expressing transcription factors.You can also have the stem cells being exposed to selected growth factors or you can culturethe stem cells along with other cell types which can induce lineage of the specific celltype.So, you can also do combinations where you treat it with growth factors or antagonistand try to use this kind of a process for differentiating cells, directing the differentiationof stem cells.So, basically this is what happens, let us look at an example where you are basicallytalking about an external signal which is the growth factor which you add.So, let us say a growth factor is present in the media in which the cells are beingcultured, what happens is this diffusible protein growth factors will bind to the receptorof the protein, will have to diffuse and reach the receptor of the surface of the proteinwhere it will bind to the receptor and then it initiates an intracellular signal whichis transmitted through protein tyrosine kinases or other intra cellular signal transducerslike G-protein and once that happens there is an action within the nucleus, which basicallyhelps in, which basically causes the expression of certain transcription factors which resultsin the cells getting differentiated ok.So, this is what you see in an external signal stimuli.So, other than this, you can also direct differentiation using biophysical properties, which wouldbe the mechanical or the physical properties of the material on which the cells are beingcultured.So, this is a brief representation of what are all the factors that can actually affect,you could have material composition could that play a role, depending on the materialcomposition how the cells interact with the material will change, if you the integrinsand cell binding, cell adhesion could all change based on the composition of the materialthat you are using.Substrate stiffness has also being shown to affect how the cells differentiate themselvesand the last one is the nanotopography were the topogra the surface topography of thematerial can actually direct cells, because that helps in while the cells are culturedthey might have to be aligned in the presence of this nanotopography and if that happensthe sense the cells will differentiate to specific cell type and so on.So, here are some examples where composition and stiffness and topography have been usedto get different types of cells.So, I am not going to go into each and everyone, but I hope you would go through this to tryand understand what each of these materials and each of these properties can actuallydo.So, this gives an overview of what people have tried and what people have been ableto accomplish.So, one last example would be alginate.Alginate is something which has been studied extensively for mesenchymal stem cells differentiationbecause they are very easy to work with and you can change their properties very easily.So, people have shown that the structure of the alginate whether it is an injectable solutionor if you have it as porous scaffold, can alter the way these molecules are deliveredthere by that can play a role in how the mesenchymal stem cells are differentiating.Another factor which has been shown to modulate differentiation is the adhesiveness of thesurface, adhesivity of alginate material.So, in the presence of a peptide, you have people have observed it to be differentiatedto osteogenic whereas, when there is no peptide it differentiate towards chondrogenic cellline even when every other property is the same.And composition can actually mediate the material properties and it can be used to convert cellssorry convert, differentiate cells to form myogenic or chondrogenic depending on whetheryou have collagen along with this or fibrin along with this.So, people have shown these kinds of differentiations are also possible.Mechanical property such as stiffness, as the stiffness is lesser you would end up formingsomething like an adipocyte and as the same stiffness increases you would end up forminga muscle or a bone tissue.So, it is quite logical right, it depends on how the ECM of the natural tissue is andbased on that this differentiation happens.So, this gives the brief introduction on the biology of differentiation and how directeddifferentiation being explored for stem cells.So, in the next lecture we will talk about little bit about the mathematical modellingaspects with respect to how this external factors interact with the surfaces and soon.Thank you.