FORM 2 THE PATENTS ACT, 1970 (39 OF 1970) & PATENTS RULES, 2006 COMPLETE SPECIFICATION (SECTION 10; RULE 13) TITLE: HYDROGEL FOR TARGETED DIFFERENTIATION OF STEM CELLS TOWARDS NEURONS Applicant : IITB MONASH RESEARCH ACADEMY Nationality: INDIAN Address : IIT Bombay Powai, Mumbai 400 076, India THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED FIELD OF INVENTION The present invention relates to the formulation containing growth factors entrapped in amyloid hydrogels for their optimal delivery in tissue engineering. In particular, the present invention relates to the use of a pH responsive amyloid hydrogel for delivery of protein based active growth factors for targeted stem cell differentiation. More particularly, the present invention relates to the methods for controlling the delivery, wherein the porosity of the mesh/matrix was altered. BACKGROUND OF INVENTION Hydrogels are a three-dimensional network made of polymers' chains (such as chemical polymers, proteins/peptides) cross-linked physically or chemically that can entrap large volume of water or biological fluid1,2. In last two decades, a significant amount of research has been done for developing various hydrogels and their potential applications. The two most potential applications of hydrogels are tissue engineering and drug delivery3"10. Hydrogels are often employed as vehicles for cell encapsulation along with suitable therapeutic drugs and growth factors to injury sites for promoting regeneration11"13. Hydrogels present a unique microstructure to the encapsulated cells influencing cell signaling and hence cellular fate. Peptide hydrogels are a class of physical hydrogels where the component peptides self-assemble non-covalently to form an ordered nanostructure14'16 and eventually supramolecular networks, which entrap water for gelation17. Recently with the advent of protein engineering, relatively simple peptide based hydrogels have gained increasing attention in biomaterial development18'21. The major advantage of employing short peptides as hydrogelators is that their properties could be easily tuned and controlled via side chain modification and chemical alteration to achieve the desired functionality22,23. Moreover, peptide hydrogels are also advantageous as they have less potential for inflammation and also have bio-degradable components 24"26 compared to polymer based hydrogels. Recently, the present inventors have shown the application of another class of hydrogels based on "amyloid fibrils" for possible application in 2D, 3D cell culture and stem cell differentiation3,4. Amyloids are highly stable protein/peptide aggregates with cross-p-sheet rich structure27. Although amyloids are historically associated with various human diseases, recent discoveries have suggested functional roles of amyloids that help organisms' survival rather than causing diseases28"31. Also, studies have shown that amyloid fibrils are rather benign or less toxic species compared to soluble oligomers32,33. Due to higher order structure, amyloid possesses unique stability against various harsh environmental conditions such as wide ranges of pH, temperature and proteases, which makes amyloids attractive for designing biomaterials for tissue engineering as well as various nanotechnological applications10,34"36. Recently, a number of studies have also indicated that amyloids are unique cell adhesive material that promotes adhesion of cells without the presence of RGD motif37,38. The inventors' recent study has also shown that amyloid based hydrogels could be suitably used as implantable materials for tissue engineering, owing to their non-inflammatory property along with ease of delivery with minimally invasive surgery4. In a natural tissue environment, extra cellular matrix (ECM) contains large depots of growth factors that affect the physiological functions of the cells13. In an attempt to mimic such a natural niche to provide better stem cell differentiation, the presence and controlled exposure of various growth factors are essential within an artificial scaffold or hydrogel. Accordingly, the present inventors have found that due to self-healing capability and stickiness towards small molecules, polymers and proteins39"41, amyloid based hydrogels are superior for growth factor loading and their controlled delivery for stem cell differentiation. Due to the higher stability and protein binding capability, amyloid hydrogels not only protect but may also release growth factors over a long period of time for stem cell differentiation. In this specification, the pH responsive amyloid based hydrogel has been demonstrated as an effective delivery vehicle for protein growth factors. This hydrogel is developed by altering the amino acid sequence of a fragment from the NAC region of the α -Syn protein, such that it self-assembles and spontaneously give a hydrogel at physiological pH. This hydrogel is thixotropic and therefore serves as an excellent material for 3D cell culture and growth factors encapsulation. This data has shown that amyloid based hydrogel can effectively entrap small fluorescence dye (FITC) and fluorescent label BSA protein and release them slowly. Further addition of salts decreases the pore size of the hydrogel and impedes the efflux of entrapped BSA, which also demonstrates that release of growth factors can also be easily modulated by the addition of salts like NaCI within the physiological limits of the human body. Based on these observations, the effect of controlled growth factors exposure was studied along with mechanical stimulation on human mesenchymal stem cells using amyloid hydrogel in 2D and 3D culture. This data has also shown that along with unique surface topography of amyloids, the stiffness of hydrogels and controlled growth factors exposure makes the amyloid based hydrogels suitable for stem cell differentiation to neuron. PRIOR ART US6281015B1 reports that 'growth factors and/or angiogenic factors are administered in combination with dissociated cells to be transplanted, preferably in microspheres with the cells on or in a polymeric matrix, to enhance survival and proliferation of the transplanted cells. Examples demonstrate that epidermal growth factor (EGF) was incorporated into microspheres fabricated from a copolymer of lactic and glycolic acid using a double emulsion technique, the incorporated EGF was steadily released over one month in vitro, and it remained biologically active, as determined by its ability to stimulate DNA synthesis, division, and long-term survival of cultured hepatocytes. EGF-containing microspheres were mixed with a suspension of hepatocytes, seeded onto porous sponges, and implanted into the mesentery of two groups of Lewis rats, to demonstrate efficacy in vivo. Two weeks after implantation in PCS animals, devices which included EGF-containing microspheres showed a two-fold increase in the number of engrafted hepatocytes, as compared to implants which received blank microspheres'. US20030187232A1 reports that 'proteins are incorporated into protein or polysaccharide matrices for use in tissue repair, regeneration and/or remodeling and/or drug delivery. The proteins can be incorporated so that they are released by degradation of the matrix, by enzymatic action and/or diffusion. As demonstrated by the examples, one method is to bind heparin to the matrix by either covalent or non-covalent methods, to form a heparin-matrix. The, heparin non-covalently binds heparin-binding growth factors to the protein matrix. Alternatively, a fusion protein can be constructed which contains a crosslinking region such as a factor Xllla substrate and the native protein sequence. It also says that incorporation of degradable linkages between the matrix and the bioactive factors can be particularly useful when long-term drug delivery is desired, for example in the case of nerve regeneration, where it is desirable to vary the rate of drug release spatially as a function of regeneration, e.g. rapidly near the living tissue interface and more slowly farther into the injury zone. Additional benefits include the lower total drug dose within the delivery system, and spatial regulation of release, which permits a greater percentage of the drug to be released at the time of greatest cellular activity'. US6451346B1 reports 'the development of pharmaceutical compositions which provide for sustained release of biologically active polypeptides. More specifically, the invention relates to the use of pH/thermosensitive biodegradable hydrogels, consisting of a A-B di block or A-B-A tri block copolymer of poly(d,l- or l-lactic acid) (PLA) or poly(lactide-co-glycolide) (PLGA) (block A) and polyethylene glycol (PEG) (block B), with ionizable functional groups on one or both ends of the polymer chains, for the sustained delivery of biologically active agents'. US6201065B1 reports 'gel-forming macromers including at least four polymeric blocks, at least two of which are hydrophobic and at least one of which is hydrophilic, and including a crosslinkable group are provided. The macromers can be covalently crosslinked to form a gel on a tissue surface in vivo. The gels formed from the macromers have a combination of properties including thermosensitivity and lipophilicity, and are useful in a variety of medical applications including drug delivery and tissue coating'. WO2013066274A1 reports 'an invention related to composite hydrogels comprising at least one non-peptidic polymer and at least one peptide having the general formula: Z-(X)m-(Y)n-Z'p, wherein Z is an N-terminal protecting group; X is, at each occurrence, independently selected from an aliphatic amino acid, an aliphatic amino acid derivative and a glycine; Y is, at each occurrence, independently selected from a polar amino acid and a polar amino acid derivative; Z' is a C-terminal protecting group; m is an integer selected from 2 to 6; n is selected from 1 or 2; and p is selected from 0 or 1. The invention further relates to methods of producing the composite hydrogels, to uses of the composite hydrogels for the delivery of drugs and other bioactive agents/moieties, as an implant or injectable agent that facilitates tissue regeneration, and as a topical agent for wound healing. The invention further relates to devices and pharmaceutical or cosmetic compositions comprising the composite hydrogels and to medical uses of the composite hydrogels'. CHALLENGES AND GAPS IN UNDERSTANDING At present, an optimal formulation in order to concomitantly provide both biochemical and bio-physical cues or neural differentiation from stem cells for obtaining a population of differentiated matured cells by targeted differentiation of stem cells is not available in the field of tissue engineering and regenerative medicine as a whole. Therefore, there is an existing need for developing an amyloid inspired scaffold from engineered peptides which is chemically well-defined and easily modifiable to suit engineering specific tissues. SUMMARY OF THE INVENTION In accordance with the present invention, there is an amyloid based scaffold material developed from a thixotropic hydrogel comprising cross-p-sheet rich structure and encapsulated with at least two protein growth factors, preferably fibroblast growth factor 8 (FGF8) and sonic hedgehog (SHH) to facilitate the stem cell differentiation towards neurons, wherein said active biochemical growth factors are released in a controlled manner by altering the porosity of the hydrogel matrix for delivery to stem cells encapsulated therein. Typically, the biopharmaceuticals and/or drugs are loaded by using said growth factor cocktail laden hydrogel during the hydrogel formation process by precisely controlling the concentration of the biopharmaceuticals and/or drugs. Typically, the hydrogel shows a distinct differentiation towards neurons for transplantation of human mesenchymal stem cells (hMSCs) into the brain by easy loading and delivery at a desired site via a minimally invasive approach. Typically, the hydrogel is used as a substrate for sustained release of the encapsulated protein growth factors altered by the salt addition for modulating the stiffness and porosity thereof. Typically, the salt addition process involves NaCI salt. Typically, the hydrogel cultures stem cells with said growth factors to be embedded in two- or three-dimensions (2D, 3D) cultures for tissue engineering applications by cell differentiation due to concomitant effect of the biophysical and bio-chemical cues obtained from said growth factor cocktail laden hydrogel. Typically, the release of encapsulated growth factors to stem cells in 3D culture is controlled by controlling the pore size via salt addition. Typically, said hydrogel is produced from amyloidogenic proteins such as alpha synuclein (a-Syn). Typically, the hydrogel encapsulated with said protein growth factors is used for controlled growth factor delivery and/or for directed stem cell differentiation to neuron. In accordance with the present invention, there is also provided a method for controlling the delivery of the formulation containing the growth factor cocktail laden hydrogel as a scaffold material as claimed in anyone of the claims 1 to 9 or a combination thereof, wherein the method comprises the following steps: a) self-assembling of the amyloid hydrogel; b) encapsulating at least two protein growth factors in said hydrogel to obtain a growth factor cocktail laden hydrogel; c) adding a salt, preferably NaCI to said growth factor cocktail laden hydrogel; and d) 2D / 3D culturing of stem cells with said growth factors embedded in the hydrogel; wherein the release or diffusion of said encapsulated growth factors is precisely controlled by reducing the pore size of said hydrogel by salt addition in the growth factor cocktail laden hydrogel. DESCRIPTION OF THE INVENTION An amyloid hydrogel encapsulating different growth factors for driving stem cell differentiation towards neurons have been reported The present invention facilitates these amyloid hydrogels to be used as a suitable and effective substrate for tissue engineering applications due to their thixotropic nature, high stability and nano-topography of amyloid fibrils. Moreover, the sustained release of the encapsulated protein molecules could also be altered by simply adding the salt, which also modulates its stiffness and porosity. The present invention also deploys the sustained exposure of growth factors from these amyloid hydrogels along with the nano-topography of thereof as an advantageous means for directed stem cell differentiation, e.g. neuronal differentiation. Since cell differentiation is the process of an unspecialized cell acquiring the cellular traits to facilitating the performance of the specialized functions, this process enables an effective differentiation of stem cells into fully functional specialized cells. Therefore, the amyloid hydrogels are used in culturing stem cells with growth factors embedded within the hydrogel in 2D, 3D for various tissue engineering applications in accordance with the present invention. The cell differentiation is attributed to the concomitant effect of the biophysical and bio-chemical cues obtained from the growth factor cocktail laden hydrogel. DETAILS OF THE INVENTION Evaluation of encapsulation and porosity modulation in amyloid hydrogel. Previous studies have shown that due to unique hydrophobic and hydrophilic surfaces, and highly ordered cross-|3-sheet structure, many small molecules, polymers and protein have shown the capability to bind to amyloids. Moreover, since amyloids are highly stable and resistant against wide variety of harsh environmental conditions such as extreme pH, temperature and proteases. The present inventors hypothesize that amyloid based materials could be potentially used as a delivery vehicle for various protein/peptide based drugs as well as growth factors in tissue engineering applications. Drug molecules and growth factors are often sensitive molecules, which may degrade if exposed to harsh environmental conditions. This sensitivity poses a threat in the encapsulation process of drugs in a hydrogel as well. Hydrogels which require chemical cross-linking, often need chemicals that may be toxic and eventually degrade the drug/therapeutic molecule loaded into it. However, aforesaid self-assembling amyloid hydrogel systems under physiological conditions are best suited for this purpose as they form hydrogel without any cross-linking agent under physiological conditions. Also, the homogeneous distribution of the encapsulated molecules is essential for uniform release. To demonstrate this, one of the pH responsive hydrogel based on α-synuclein protein sequence reported earlier by the present inventors is selected. This hydrogel is composed of a self-assembling peptide with the sequence Fmoc-VHAVA-COOH and henceforth termed A8. This class of hydrogels being thixotropic can easily encapsulate and provide sustained release of growth factors for stem cell differentiation. Hydrogel A8 does not require any addition of salt for the formation of self-sustaining hydrogel (Figure. 1). However, if 150 mM NaCI is added to the hydrogel during the induction of gelation, a much stiffer hydrogel was observed with the same volume of the hydrogel. Storage and loss modulus of the hydrogel A8 in the presence and absence of NaCI were calculated from rheological measurements with a frequency sweep. The storage modulus of the hydrogel changes from -390 Pa to -530 Pa due to the addition of the salt, as seen from the rheology measurements (Figure. 2A and 2B). Previous reports suggest a strong co-relation between hydrogel stiffness and porosity. Thus, besides the storage modulus, the pore size of the hydrogels was also quantified from the images obtained via cryo-SEM. Cryo-SEM has been used earlier by different investigators for measurement of pore sizes in the hydrogel. Consistent with earlier reports, an inverse correlation of gel porosity and gel stiffness was also observed. Hydrogel A8+NaCI had the average pore size 4 µm compared to A8 only with average pore diameter 10 µm (Figure. 2C and 2D). To study the possibility of growth factor delivery for stem cell differentiation, first the diffusion of small fluorescent dye FITC and FITC labelled BSA encapsulated in the hydrogel A8 was studied. Encapsulation of the fluorescent molecules was done by harnessing the thixotropic behavior of the hydrogel A8. A step-strain oscillatory rheology was performed to demonstrate the self-healing nature of the hydrogel (Figure. 3A and 3B). The hydrogel network is first disrupted with application of high strain (100%) and then allowed to recover under low strain (0.05%) for three cycles. Under high strain and hydrogel disruption, the storage modulus (G') drops below the loss modulus (G") indicating a fluid like or "sol" behaviour. On removal of this high strain in the subsequent step (0.05% strain), the hydrogel quickly recovers to the "gel" state as indicated by the increment of G' over G". To mix the dye/protein molecules, A8 gel was briefly vortexed and the dye was quickly mixed. Subsequently, the hydrogel was allowed to recover for 20 minutes and then studied for release of the encapsulated dye over time. Study of diffusion of small molecules and proteins from the hydrogel The interactions between the fluorescent probes and the amyloid fibrils in the hydrogel network would affect the overall diffusivity of the molecules from the hydrogel. Also, during the construction of 3D systems to help differentiation of encapsulated cells, it is important to analyze the self-diffusion of the growth factors within the hydrogel. To understand this property, the fluorescence recovery was performed after photobleaching (FRAP) experiments with hydrogels encapsulating FITC and FITC-BSA. Earlier reports suggested FRAP as a convenient and reliable method to understand the self-diffusion coefficient in hydrogels and also the matrix-probe interaction during the course of diffusion. For this, fluorescent probe FITC and FITC-BSA were homogeneously mixed with the hydrogel A8 and a high intensity laser was used to photo-bleach a particular area in the hydrogel of radius 8 µm (Figure. 3C). After bleaching, the laser power was attenuated and the recovery of fluorescence was captured. The recovery of the fluorescence was due to diffusion of the non-bleached fluorescent probes from adjacent areas to the photobleached area (Figure. 3C). This was measured as a function of time and the resultant data was fitted to determine the diffusion coefficient of the probe within the network of the hydrogel. For hydrogel A8 mixed with FITC, the individual stages of recovery are marked from 1-4 (Figure. 3C), with stage 1 showing the pre-bleached image of the hydrogel. Stage 2 represents 50% photobleaching after which the recovery was captured (stage 3-4) at 1% laser power. The fluorescence recovery data was normalized and plotted with an increase in time according to equation 3 (see method section). The half-life of recovery for FITC in A8 hydrogel was calculated as 63s with a diffusion coefficient of 25x10"B cm2/s. Identical experiments were performed with FITC-BSA for A8 and A8+NaCI hydrogel (Figure. 3D). As calculated from FRAP experiments, half-life of recovery T 1/2 (s) for FITC-BSA was 150s and 190s, respectively for A8 and A8+NaCI hydrogel (Figure. 3E). The self-diffusion coefficient D (10'8 cmz/s) for A8 and A8+NaCI was 10.6 cmz/s and 8 cm2/s (Figure. 3F), respectively. The data indicates that movement of the encapsulated protein is slower in A8+NaCI hydrogel, compared to A8 only resulting in a slower diffusion. The fluorescence recovery of the same probe increases from the buffer to A8 to A8+NaCI, indicating that mobility of the protein is more restricted within the hydrogel when the porosity decreases. The data indicates that the release of encapsulated growth factors could be impeded by reducing the pore size via salt addition. This would ensure sustained exposure of the growth factors to the stem cells in 3D culture. The decrease in the mobility of the probe in A8 gels in presence of NaCI may be due to the smaller mesh size of the A8+NaCI hydrogel compared to A8 alone. However, there may be an additional probe-matrix interaction that affects the macro-molecular mobility of the fluorescent probes within the amyloid fibril network of the hydrogel. Bulk release studies Although the movement of the tracer dye or fluorescent labelled protein within the 3D meshwork of the hydrogel could be estimated via FRAP, the bulk release of the entrapped molecules would help in understanding their release in a 2D culture due to macromolecular diffusion as well as surface erosion of the hydrogel. For optimal differentiation, the growth factors should be encapsulated for a longer time period in the hydrogel network and released in a sustained fashion. To study and model the bulk release of the encapsulated proteins in the hydrogel, FITC labelled BSA was encapsulated within A8 hydrogels prepared in presence and absence of 150 mM NaCI as described in FRAP experiment. In the study, FITC-BSA was encapsulated in A8 and A8+NaCI hydrogel by vortexing the hydrogel and promptly mixing the desired amount of FITC-BSA (final concentration 1000 u.g/ml_) in the hydrogel. After loading, the hydrogels were allowed to recover for 16-24 hours. Then, 300 µL of phosphate buffer was layered on the top of the hydrogel to probe the release of the tracer from the hydrogel meshwork. The entrapped fluorescent protein would diffuse out slowly into the bulk solution from the hydrogel. The released FITC-BSA was tracked via fluorescence spectroscopy in a 96 well plate. Figure 3G shows the fluorescence from the amount of FITC-BSA released from the hydrogels at different time points (5) to the amount encapsulated initially (F0) over a period of 30 days. As evident from Figure 3G, the amount of FITC labelled BSA released was different from A8 hydrogel formed in presence and absence of NaCI. The inset shows release kinetics of FITC-BSA for the initial 15 hours for both A8 and A8+NaCI. A much more sustained release profile of the encapsulated BSA was observed from the gel formed in presence of NaCI. The time taken for the release of 50% of the initial amount of encapsulated fluorescent protein, t50% is 14 days for A8+NaCI hydrogel compared to only 6 days for A8 hydrogel. This characteristic release time is also consistent with the trend of FRAP data (Figure. 3D). The experiment also shows that more amount of FITC-BSA is released from A8 hydrogel compared to A8+NaCI set under the study period of 30 days. For A8 hydrogel, 95% of the initial encapsulated protein got released, whereas for A8+NaCI, only 79% got released after 30 days of incubation (Fig. 3G; end-point). This difference couid be attributed to a smaller pore size of the hydrogel A8+NaCI, where bulk diffusion of the encapsulated molecule would be impeded. The data reflects that a small amount of salt addition under physiological limits alters such a drastic release profile of encapsulated protein. It should be worth mentioning here that during the course of measurement, surface erosion and gradual degradation was not found in any of the hydrogels, Although the overall integrity of both the hydrogels (A8 alone and A8+NaCI) was almost the same from visual inspection, surface erosion to a minor extent could not be ruled out. Thus, the release of BSA could be attributed mostly to diffusion of FITC-BSA through the hydrogel network. Effect of growth factors encapsulation on structure and morphology of amyloid hydrogels. Amyloid fibrils possess distinct characteristics in terms of its bio-physical properties. A typical feature of amyloid fibril is the presence of cross-p sheet structure, which is reflected in Fourier transform infrared spectroscopy with distinct peaks centered at ~1630 cm-1 and ~1690 cm"1 and appearance as nano-fibers under the electron microscope. These nano-fibrils are generally ~6-12 nm in diameter and a few microns in length42. This motif comprising of tightly interacting cross-β sheets among the interacting proteins provides stability to amyloid fibrils. The addition of growth factors to the hydrogel may alter the fibril morphology or structural integrity of the component nano-fibers. To rule out such possibilities, FTIR and TEM studies were performed to observe possible changes in the amyloid fibrils pre- and post-encapsulation of growth factors. FTIR peaks corresponding to amide-l absorbance were used to examine the secondary structure of protein43,44. For FTIR spectra measurement, both the gel A8 and A8+gf, were vortexed, layered on KBr pellet and dried below the IR lamp. Thereafter, FTIR absorbance spectra of both the samples was acquired in the range of 1500 cm-1 to 1800 cm-1 and the region corresponding to amide-l absorbance (1600 cm-1 to 1700 cm-1) was deconvoluted and curve fitted in Opus 65 software according to the manufacturer's instruction. FTIR spectra of gel formed from A8 peptide show intense peaks at 1624 cm'1, 1631 cm"1, 1637 cm-1 and 1689 cm-1, indicating the presence of antiparallel p-sheet structure (Fig.4A). Identical peaks were also observed for A8 hydrogel with encapsulated growth factors (A8+gf) (Figure. 4A). The overall results have shown that there is not any significant difference in the secondary structure of A8 gel formed in absence and presence of growth factors. EM images reveal that fibrils within the hydrogel had identical morphologies (Figure. 4B) and diameter (Figure. 4C) whether or not the growth factors FGF8 and SHH were encapsulated. Thus, the interaction of the growth factors with the fibrils of A8 hydrogel did not alter them morphologically or structurally. Growth factor delivery via A8 hydrogel formed in presence and absence of NaCI in 2D of hMSCs Bone marrow derived stem cells are capable of differentiating into cells of different lineages of mesenchymal origin like chondrocytes, osteocytes etc. However, they are also capable of differentiating into non-mesenchymal cells like neural cells under suitable conditions. Earlier, differentiation of hMSCs was tried and carried out by the addition of various growth factors in the culture media at different stages of growth or maturation. However, these mechano-sensitive cells also respond to substrate stiffness and different groups have shown that stem cells preferably differentiate towards neuronal lineage when cultured on soft substrate. More recently, it was discovered that coordinated interaction between the stem cells, soluble bio-chemical growth factors and substrate stiffness dictate the fate of these cells, In pursuit to mimic this dynamic niche for human mesenchymal stem cells (hMSCs), amyloid hydrogels have been used encapsulated with various growth factors required for trans-differentiation of hMSCs to neuronal lineage. For this, hMSCs both in 2D and 3D on A8 hydrogel matrix preloaded with fibroblast growth factor 8 (FGF8) and sonic hedgehog (SHH) have been cultured. These growth factors are known to help in differentiation of hMSCs towards neurons. For 2D culture, A8 hydrogel was vortexed and mixed with growth factors. Subsequently, it was cast on a treated cover glass (12 mm diameter) and allowed to reform into the hydrogel for 20 minutes in a bio-safety hood. hMSCs were then seeded on the hydrogel and cultured in proliferation media (Knockout DMEM with 10% FBS)for 24 hours. Around 5,000 cells were seeded per well in a 24 well plate. After 24 hours of incubation, the media was replaced with Neurobasal-A media supplemented with B27. As controls, hMSCs were cultured on A8 hydrogel and cover glass only. To study the controlled exposure versus constant presence of growth factor in media, hMSCs were also cultured on glass with growth factor (gf) added to the media. Cells were imaged under phase contrast microscope after 14 days of culture (Figure. 5A). The morphology of the cultured cells was analyzed by quantifying cell spread area and circularity using ImageJ software. Generally, cell circularity gives a good estimate of how elongated the cells are. Neuronal cells tend to have an elongated bipolar morphology, which is reflected in lower cell circularity. If the cell shape is fitted inside an ellipse, the ratio of the minor to major axis reflects cell circularity. When the cell is rounded, circularity value approaches unity, whereas more they elongate along the major axis, the circularity value decreases. hMSCs were much elongated and polarized on A8 and A8+gf set compared to those cultured on glass. When the cells were cultured on glass, which was exposed to growth factors via culture media, they also showed bipolar morphology, but with smaller in size (Figure. 5B) and less branched (Fig. 5C) compared to cells cultured on A8 encapsulated with growth factors, as evident from the cell spread area and circularity quantification. Overall, the cells cultured on glass coverslips occupied largest spread area followed by cells on A8, A8+NaCI +gf, A8+NaCI, A8+gf and gf only (Fig. 5B). The cell spread area and cell circularity were also significantly different between glass and all other substrates. The cells cultured on hydrogel formed in presence of NaCI and growth factors (A8+NaCI+gf), where growth factor (gf) may be released in the most sustained fashion, show the most branched morphology at the end of 14 days of culture. This data suggests that cells assume much more neuron like morphology when they are exposed concomitantly to both physical cues via amyloid hydrogel and bio-chemical cues via the encapsulated growth factors. When cultured cells were fixed at the end of 14 days' culture and immunostained for neuronal marker |3-IH tubulin (Figure. 6A) and an increased expression of β-III tubulin was observed for cells cultured on hydrogel in presence and absence of growth factors compared to cells cultured on glass under identical conditions. The expression of β-III tubulin in the cells cultured on different substrates were quantified from the fluorescent images by ImageJ (Figure. 6B). Here again, the stem cells cultured on A8+NaCI+gf substrate showed significantly high expression of (3-HI tubulin, followed by A8+NaCI, A8 and A8+gf compared to the glass substrate. The control cells cultured on glass with growth factors in the media also showed higher |3-lll tubulin expression than cells on glass, but lower than those cultured on hydrogels. Since the effect of neuronal differentiation on soft amyloid hydrogels are attributed to mechano-transduction3,4, alteration of cytoskeletal rearrangements was checked with staining of actin network of the cells cultured under different conditions (Fig. 7). The actin fibers of cells cultured on glass showed to form bundles of stress fibers spanning across the cells (Fig. 7A). Whereas in cells cultured on A8 hydrogel formed in presence and absence of NaCI with growth factor encapsulation, actin fibers were localized at the edges of the protrusions of cells and strong bundles of stress fibers were absent (Fig. 7C and Fig. 7D respectively). This kind of actin rearrangement is reported during differentiation of stem cells45. The thick actin fibers were also missing when cells were cultured with growth factors present in the media (Figure. 7B). Therefore, the data suggests that soft substrate, surface topography of amyloid and presence of growth factors in a sustained manner may help to differentiate stem cells towards neuron. Growth factors delivery in 3D culture for stem cell differentiation Traditionally culture of mammalian cells is done in 2D on tissue culture treated polystyrene or on variety of other substrates. Study on this 2D culture has led to several seminal findings on how cells interact with the substrate and how cellular responses are modulated via cell-substrate interaction. However, recent studies indicate that assessment of the effects of epigenetic factors on cells might not be reflected in 2D cell culture, as these often exhibit unnatural behavior when cultured in monolayers compared to their native 3D tissues46. Based on the above facts, it was hypothesized that 3D culture of stem cells with hydrogel encapsulating growth factors might help in their better differentiation. Also, prolonged retention of exogenously added growth factors are more desirable, as cells would be exposed to the active growth factors for a longer duration during its differentiation phase. For this, hMSCs were cultured in 3D with A8 hydrogel, A8 with encapsulated growth factors