 I am from Indian Institute of Technology, Madras. It is located in the southern part of India. And it's known for its bad summer. So today, I would like to speak about a scaffold-based approach for neural lineage differentiation of human embryonic stencils. So embryonic stencils. These are basically pluripotent in nature, which means they can self-renew pain definitely, and they can become into any type of cell. So you can have like electrodes or cardiomyocytes or neural cells. So they are basically a limitless supply of cells. So what are the uses of embryonic stencils? They can be used for screening various kinds of drugs. You can understand the human development process. But the most important application comes in the cell transplantation therapy. Some systems in the human body, like for example, the adult neural system, is incapable of healing itself for a patient under more certain disease or disorder. He's left with motor impairment or paralysis or other malfunctioning. So the theoretical idea is if you can create neurological cells from the stem cells and you can transplant them into the patient to restore the normal function. But so far, it has not been widely possible as such that there have been only two or three clinical trials so far. This is mainly because of the traditional, the embryonic stem cell culture conditions are not chemically well defined. For clinical applications, you need well defined culture conditions. Because if you even have a single non-target cell in your body, you can have aberrant tissue growths. So for differentiating the human embryonic stem cells into particular type of cells, but most of these have a lot of disadvantages, like they can contaminations or they can embryo body formation as an example. So basically, when the stem cells don't have a surface to adhere upon, they form aggregates or lumps. These are basically the embryo bodies. So when these are left to spontaneously differentiate, they differentiate into all the three germ layers. So what is usually that is certain small molecules like redinoic acid is added in the culture to better the differentiation of these embryonic bodies into particular lineage. So this particular case, redinoic acid is used for the detection of neural differentiation. So as you can see, since in an embryo, there is highly oriented differentiation. But in this case, since it is in a 3D manner, you can have it's highly disorganized. And you have a lot of batch to batch variations. And it all depends upon the embryo body size, cell density, and the diffusion of the soluble factor, all these come into play. So this is not particularly a reliable method. So why is it so difficult for differentiating these cells into a particular cell type? Why are there so many constraints? If you see in the embryo, in order to maintain the flowable density in nature or in order to determine a body model's cell fate, a lot of factors come into play. Like you have cell-cell interactions. You have the interactions of soluble factors in the medium. And most of them have the cell and matrix interactions. If you see, a lot of developments have been done when you take the soluble factors or when you take the neighboring cell, controlling the cell-cell interactions. But the substrates are highly developed. For example, if you see, the partial tissues have elasticity in a range like 1 kilopascal to 100 kilopascal. But when you see the traditional tissue culture materials, it's completely out of range. So the answer for this is hydrogels. And hydrogels are basically a network of hydrophilic polymers. Recently, a lot of focus has been on these as catapults force and load. So basically, these have tissue-length physical and mechanical properties. These are basically the polymers dispersed in water. So hydrogels, we use hydrogels also for various reasons. Like the first form of reason is you can control the mechanical property of the hydrogel. And it is also in the range of the physiological tissue. So for example, if you change the cross-linking density, you can change the elasticity. And you can have different ligands. And you can have different ligand densities. And you can have multiple ligands. So there are different types of hydrogels. Like, for example, when you use a polyanionic cation, you have basically ionic hydrogels. And you also have chemical hydrogen and physical hydrogen. Basically, you have a hydrophobic polymer in which you insert polar groups by various reactions like hydrolysis, oxidation, or sulfonation. And if these are like, we mean together as network only through hydrophobic interactions, then it is called a physical hydrogen. Or if you cross-link them, and basically covalent bonds are formed, it is known as a chemical hydrogen. We used chemical hydrogels because when we are inserting the peptide on hydrogel, basically the ligand on the hydrogel, the ligands are charged. And we don't want any unnecessary electrostatic interactions. So we prefer the chemical hydrogels. So how do we go about synthesizing these hydrogels? First, we start off with a glass coverslip. We functionalize the coverslip with reactive groups. This is basically for covalently binding the hydrogen on the coverslip so that it is easy to handle throughout the reaction. Then we go on forming the hydrogen. Basically, we use this acrylamide and acrylamide. And we use some polymerizing agents. And then we functionalize it with acrylic N-hedges. Now this acrylic N-hedges basically reacts with the amine groups. So the peptides can have multiple amine groups. We don't want these peptides to attach the hydrogen any fashion they want. We want to control the way they attach to the peptides. So we follow another method. Instead of using these acrylic N-hedges for getting the peptides to bind on them, we again reacted these, but ducomyne and aminomalamide. Basically, this aminomalamide reacts with the thiol group. So what we do is we take the peptide of our interest. And we insert a sustained residue in the peptide in either the C-terminal or N-terminal, depending on the manner in which the cell interacts with the peptide. So since our thiol group, it reacts with it. And we use ducomyne because ducomyne interacts with the cell, not interacts with the peptide. So basically, we use it for controlling the cell density which is adhering on the hydrogen. So if we change the ratio of ducomyne and aminomalamide, the honey cell is adhered on the hydrogels. So we change the elasticity by changing the ratio of busaprylamide and acrylamide. And we can have different peptides attached. So by following this method, we prepare hydrogels with different elasticity. And as you can see, the aminomaline stem cells basically show different behavior on hydrogels with different elasticity. The hydrogels with the elasticity 10-hilo pascal basically support the aminomaline stem cell pluripotent nature, like for it. So apparently an aminomaline stem cell is not a company. So you can see that these, there are many cells attached to this hydrogel and up to 60, even up to 60 days or so, we pass each every four to seven days. That is basically, this is the immunostaining of the cells. And that basically stains the nucleus and these are pluripotent markers. So you can see the cells are attached in colonies over here. We can see the colony as such, but the cells are in colonies. And they're expressing the markers of pluripotency. But if you see the other two cases, there are very few cells attached in these. All the other conditions are the same, only the elasticity was changed, but you can see the different behavior of the cells towards the scaffold. So basically, if you observe the brain tissue's elasticity, like it corresponds to this and you have different biological tissues have different elasticity. So we thought that since the embryonic stem cells responding to the elasticity of the matrix and you have different biological tissues with different elasticity. So our hypothesis of the big question is, can the matrix elasticity basically direct the embryonic stem cell differentiation? So in order to answer this, first we set, first we chose a cell line and we actually made sure that it gives a heterogeneous population and not, it doesn't give up your neural population. So you can see different cells here. These don't have any projections. And then what we did, we took this 0.7 kilopascal hydrogen. If you remember that this is the brain tissue's elasticity. So when this was added to different shape, you can see they are in colonies over here and they're slowly starting to move away from the colonies as they differentiate. And we can see they showed projections from the projections are coming from the cell. And we wanted to come forward by doing a neurostaining. So we used this Tajwan marker. Basically this beta tubulin is a hydrotubule element. It is present exclusively in the neurons. So we signed this and we observed that it did express the neuronal marker. So this was very interesting, but of course we need to do a lot more functional assays and other quantification methods to confirm this. So if this is like improved and made more efficient, it can have a lot of impact in the regenerative medicine. So to summarize, we need proper well-defined condition, we need efficient reproducible methods of clinical application of these embryonic stem cells. And we saw that these embryonic stem cells correspond to the mechanical cues from the scaffold. We know that the different biological tissues have different elasticities. And therefore, and we did observe that the cells differentiated on different elasticity which show different behavior. And the particular keys express the neuronal marker. So that's what I've been doing all summer. So, acknowledgements. I would like to acknowledge Professor Lara Kisling for hosting me and for being a source of inspiration. And I would like to thank Samira Musa for guiding me throughout the project. And I would like to thank all the Kisling lab members for encouraging me and being so friendly and making it as almost home for me. And I would like to thank Karana Program, Iris, as a city of DBT for funding. Thank you.