Mitotic outcomes and errors in fibrous environments.

During mitosis, cells round up and utilize the interphase adhesion sites within the fibrous extracellular matrix (ECM) as guidance cues to orient the mitotic spindles. Here, using suspended ECM-mimicking nanofiber networks, we explore mitotic outcomes and error distribution for various interphase cell shapes. Elongated cells attached to single fibers through two focal adhesion clusters (FACs) at their extremities result in perfect spherical mitotic cell bodies that undergo significant 3-dimensional (3D) displacement while being held by retraction fibers (RFs). Increasing the number of parallel fibers increases FACs and retraction fiber-driven stability, leading to reduced 3D cell body movement, metaphase plate rotations, increased interkinetochore distances, and significantly faster division times. Interestingly, interphase kite shapes on a crosshatch pattern of four fibers undergo mitosis resembling single-fiber outcomes due to rounded bodies being primarily held in position by RFs from two perpendicular suspended fibers. We develop a cortex-astral microtubule analytical model to capture the retraction fiber dependence of the metaphase plate rotations. We observe that reduced orientational stability, on single fibers, results in increased monopolar mitotic defects, while multipolar defects become dominant as the number of adhered fibers increases. We use a stochastic Monte Carlo simulation of centrosome, chromosome, and membrane interactions to explain the relationship between the observed propensity of monopolar and multipolar defects and the geometry of RFs. Overall, we establish that while bipolar mitosis is robust in fibrous environments, the nature of division errors in fibrous microenvironments is governed by interphase cell shapes and adhesion geometries.

Dendritic cell force-migration coupling on aligned fiber networks.

Dendritic cells (DCs) are antigen-presenting cells that reside in peripheral tissues and are responsible for initiating adaptive immune responses. As gatekeepers of the immune system, DCs need to continuously explore their surroundings, for which they can rapidly move through various types of connective tissue and basement membranes. DC motility has been extensively studied on flat 2D surfaces, yet the influences of a contextual 3D fibrous environment still need to be described. Using ECM-mimicking suspended fiber networks, we show how immature DCs (iDCs) engage in migratory cycles that allow them to transition from persistent migration to slow migratory states. For a subset of iDCs with high migratory potential, we report the organization of protrusions at the front of the cell body, which reverses upon treatment with inflammation agent PGE2. We identify an unusual migratory response to aligned fiber networks, whereby iDCs use filamentous protrusions to attach laterally and exert forces on fibers to migrate independent of fiber alignment. Increasing the fiber diameter from 200 nm to 500 nm does not significantly affect the migratory response, however, iDCs respond by forming denser actin bundles around larger diameters. Overall, the correlation between force-coupling and random migration of iDCs in aligned fibrous topography offers new insights into how iDCs might move in fibrous environments in vivo.

Rules of contact inhibition of locomotion for cells on suspended nanofibers.

Contact inhibition of locomotion (CIL), in which cells repolarize and move away from contact, is now established as a fundamental driving force in development, repair, and disease biology. Much of what we know of CIL stems from studies on two-dimensional (2D) substrates that do not provide an essential biophysical cue-the curvature of extracellular matrix fibers. We discover rules controlling outcomes of cell-cell collisions on suspended nanofibers and show them to be profoundly different from the stereotyped CIL behavior on 2D substrates. Two approaching cells attached to a single fiber do not repolarize upon contact but rather usually migrate past one another. Fiber geometry modulates this behavior; when cells attach to two fibers, reducing their freedom to reorient, only one cell repolarizes on contact, leading to the cell pair migrating as a single unit. CIL outcomes also change when one cell has recently divided and moves with high speed-cells more frequently walk past each other. Our computational model of CIL in fiber geometries reproduces the core qualitative results of the experiments robustly to model parameters. Our model shows that the increased speed of postdivision cells may be sufficient to explain their increased walk-past rate. We also identify cell-cell adhesion as a key mediator of collision outcomes. Our results suggest that characterizing cell-cell interactions on flat substrates, channels, or micropatterns is not sufficient to predict interactions in a matrix-the geometry of the fiber can generate entirely new behaviors.

Bioenergetics underlying single-cell migration on aligned nanofiber scaffolds.

Cell migration is centrally involved in a myriad of physiological processes, including morphogenesis, wound healing, tissue repair, and metastatic growth. The bioenergetics that underlie migratory behavior are not fully understood, in part because of variations in cell culture media and utilization of experimental cell culture systems that do not model physiological connective extracellular fibrous networks. In this study, we evaluated the bioenergetics of C2C12 myoblast migration and force production on fibronectin-coated nanofiber scaffolds of controlled diameter and alignment, fabricated using a nonelectrospinning spinneret-based tunable engineered parameters (STEP) platform. The contribution of various metabolic pathways to cellular migration was determined using inhibitors of cellular respiration, ATP synthesis, glycolysis, or glucose uptake. Despite immediate effects on oxygen consumption, mitochondrial inhibition only modestly reduced cell migration velocity, whereas inhibitors of glycolysis and cellular glucose uptake led to striking decreases in migration. The migratory metabolic sensitivity was modifiable based on the substrates present in cell culture media. Cells cultured in galactose (instead of glucose) showed substantial migratory sensitivity to mitochondrial inhibition. We used nanonet force microscopy to determine the bioenergetic factors responsible for single-cell force production and observed that neither mitochondrial nor glycolytic inhibition altered single-cell force production. These data suggest that myoblast migration is heavily reliant on glycolysis in cells grown in conventional media. These studies have wide-ranging implications for the causes, consequences, and putative therapeutic treatments aimed at cellular migration.

Crosshatch nanofiber networks of tunable interfiber spacing induce plasticity in cell migration and cytoskeletal response.

Biomechanical cues within tissue microenvironments are critical for maintaining homeostasis, and their disruption can contribute to malignant transformation and metastasis. Once transformed, metastatic cancer cells can migrate persistently by adapting (plasticity) to changes in the local fibrous extracellular matrix, and current strategies to recapitulate persistent migration rely exclusively on the use of aligned geometries. Here, the controlled interfiber spacing in suspended crosshatch networks of nanofibers induces cells to exhibit plasticity in migratory behavior (persistent and random) and the associated cytoskeletal arrangement. At dense spacing (3 and 6 µm), unexpectedly, elongated cells migrate persistently (in 1 dimension) at high speeds in 3-dimensional shapes with thick nuclei, and short focal adhesion cluster (FAC) lengths. With increased spacing (18 and 36 µm), cells attain 2-dimensional morphologies, have flattened nuclei and longer FACs, and migrate randomly by rapidly detaching their trailing edges that strain the nuclei by ∼35%. At 54-µm spacing, kite-shaped cells become near stationary. Poorly developed filamentous actin stress fibers are found only in cells on 3-µm networks. Gene-expression profiling shows a decrease in transcriptional potential and a differential up-regulation of metabolic pathways. The consistency in observed phenotypes across cell lines supports using this platform to dissect hallmarks of plasticity in migration in vitro.-Jana, A., Nookaew, I., Singh, J., Behkam, B., Franco, A. T., Nain, A. S. Crosshatch nanofiber networks of tunable interfiber spacing induce plasticity in cell migration and cytoskeletal response.

Nanonet Force Microscopy for Measuring Cell Forces.

The influence of physical forces exerted by or felt by cells on cell shape, migration, and cytoskeleton arrangement is now widely acknowledged and hypothesized to occur due to modulation of cellular inside-out forces in response to changes in the external fibrous environment (outside-in). Our previous work using the non-electrospinning Spinneret-based Tunable Engineered Parameters' suspended fibers has revealed that cells are able to sense and respond to changes in fiber curvature and structural stiffness as evidenced by alterations to focal adhesion cluster lengths. Here, we present the development and application of a suspended nanonet platform for measuring C2C12 mouse myoblast forces attached to fibers of three diameters (250, 400, and 800 nm) representing a wide range of structural stiffness (3-50 nN/µm). The nanonet force microscopy platform measures cell adhesion forces in response to symmetric and asymmetric external perturbation in single and cyclic modes. We find that contractility-based, inside-out forces are evenly distributed at the edges of the cell, and that forces are dependent on fiber structural stiffness. Additionally, external perturbation in symmetric and asymmetric modes biases cell-fiber failure location without affecting the outside-in forces of cell-fiber adhesion. We then extend the platform to measure forces of (1) cell-cell junctions, (2) single cells undergoing cyclic perturbation in the presence of drugs, and (3) cancerous single-cells transitioning from a blebbing to a pseudopodial morphology.

Role of suspended fiber structural stiffness and curvature on single-cell migration, nucleus shape, and focal-adhesion-cluster length.

It has been shown that cellular migration, persistence, and associated cytoskeletal arrangement are highly dependent on substrate stiffness (modulus: N/m(2) and independent of geometry), but little is known on how cells respond to subtle changes in local geometry and structural stiffness (N/m). Here, using fibers of varying diameter (400, 700, and 1200 nm) and length (1 and 2 mm) deposited over hollow substrates, we demonstrate that single mouse C2C12 cells attached to single suspended fibers form spindle morphologies that are sensitive to fiber mechanical properties. Over a wide range of increasing structural stiffness (2 to 100+ mN/m), cells exhibited decreases in migration speed and average nucleus shape index of ∼57% (from 58 to 25 µm/h) and ∼26% (from 0.78 to 0.58), respectively, whereas the average paxillin focal-adhesion-cluster (FAC, formed at poles) length increased by ∼38% (from 8 to 11 µm). Furthermore, the increase in structural stiffness directly correlates with cellular persistence, with 60% of cells moving in the direction of increasing structural stiffness. At similar average structural stiffness (25 ± 5 mN/m), cells put out longer FAC lengths on smaller diameters, suggesting a conservation of FAC area, and also exhibited higher nucleus shape index and migration speeds on larger-diameter fibers. Interestingly, cells were observed to deform fibers locally or globally through forces applied through the FAC sites and cells undergoing mitosis were found to be attached to the FAC sites by single filamentous tethers. These varied reactions have implications in developmental and disease biology models as they describe a strong dependence of cellular behavior on the cell's immediate mechanistic environment arising from alignment and geometry of fibers.

Suspended micro/nanofiber hierarchical biological scaffolds fabricated using non-electrospinning STEP technique.

Extracellular matrix (ECM) is a fibrous natural cell environment, possessing complicated micro- and nanoarchitectures, which provide extracellular signaling cues and influence cell behaviors. Mimicking this three-dimensional microenvironment in vitro is a challenge in developmental and disease biology. Here, suspended multilayer hierarchical nanofiber assemblies (diameter from micrometers to less than 100 nm) with accurately controlled fiber orientation and spacing are demonstrated as biological scaffolds fabricated using the non-electrospinning STEP (Spinneret based Tunable Engineered Parameter) fiber manufacturing technique. Micro/nanofiber arrays were manufactured with high parallelism (relative angles between fibers were maintained less than 6°) and well controlled interfiber spacing (<15%). Using these controls, we demonstrate a bottom up hierarchical assembly of suspended six layer structures of progressively reduced diameters and spacing from several polymer systems. We then demonstrate use of STEP scaffolds to study single and multicell arrangement at high magnifications. Specifically, using double layer divergent (0°-90°) suspended nanofibers assemblies, we show precise quantitative control of cell geometry (change in shape index from 0.15 to 0.57 at similar cell areas), and through design of scaffold porosity (80 × 80 µm(2) to 5 × 5 µm(2)) quadruple the cell attachment density. Furthermore, using unidirectional or crisscross patterns of sparse and dense fiber arrays, we are able to control the cell spread area from ∼400 to ∼700 µm(2), while the nucleus shape index increases from 0.75 to 0.99 with cells nearly doubling their focal adhesion cluster lengths (∼15 µm) on widely spaced nanofiber arrays. The platform developed in this study allows a wide parametric investigation of biophysical cues which influence cell behaviors with implications in tissue engineering, developmental biology, and disease biology.

Confinement in fibrous environments positions and orients mitotic spindles.

Accurate positioning of the mitotic spindle within the rounded cell body is critical to physiological maintenance. Adherent mitotic cells encounter confinement from neighboring cells or the extracellular matrix (ECM), which can cause rotation of mitotic spindles and, consequently, titling of the metaphase plate (MP). To understand the positioning and orientation of mitotic spindles under confinement by fibers (ECM-confinement), we use flexible ECM-mimicking nanofibers that allow natural rounding of the cell body while confining it to differing levels. Rounded mitotic bodies are anchored in place by actin retraction fibers (RFs) originating from adhesion clusters on the ECM-mimicking fibers. We discover the extent of ECM-confinement patterns RFs in 3D: triangular and band-like at low and high confinement, respectively. A stochastic Monte-Carlo simulation of the centrosome (CS), chromosome (CH), membrane interactions, and 3D arrangement of RFs on the mitotic body recovers MP tilting trends observed experimentally. Our mechanistic analysis reveals that the 3D shape of RFs is the primary driver of the MP rotation. Under high ECM-confinement, the fibers can mechanically pinch the cortex, causing the MP to have localized deformations at contact sites with fibers. Interestingly, high ECM-confinement leads to low and high MP tilts, which mechanistically depend upon the extent of cortical deformation, RF patterning, and MP position. We identify that cortical deformation and RFs work in tandem to limit MP tilt, while asymmetric positioning of MP leads to high tilts. Overall, we provide fundamental insights into how mitosis may proceed in fibrous ECM-confining microenvironments in vivo.

Ultra-thin and ultra-porous nanofiber networks as a basement-membrane mimic.

Current basement membrane (BM) mimics used for modeling endothelial and epithelial barriers in vitro do not faithfully recapitulate key in vivo physiological properties such as BM thickness, porosity, stiffness, and fibrous composition. Here, we use networks of precisely arranged nanofibers to form ultra-thin (∼3 µm thick) and ultra-porous (∼90%) BM mimics for blood-brain barrier modeling. We show that these nanofiber networks enable close contact between endothelial monolayers and pericytes across the membrane, which are known to regulate barrier tightness. Cytoskeletal staining and transendothelial electrical resistance (TEER) measurements reveal barrier formation on nanofiber membranes integrated within microfluidic devices and transwell inserts. Further, significantly higher TEER values indicate a biological benefit for co-cultures formed on the ultra-thin nanofiber membranes. Our BM mimic overcomes critical technological challenges in forming co-cultures that are in proximity and facilitate cell-cell contact, while still being constrained to their respective sides. We anticipate that our nanofiber networks will find applications in drug discovery, cell migration, and barrier dysfunction studies.

Engineering high post-electroporation viabilities and transfection efficiencies for elongated cells on suspended nanofiber networks.

The impact of cell shape on cell membrane permeabilization by pulsed electric fields is not fully understood. For certain applications, cell survival and recovery post-treatment is either desirable, as in gene transfection, electrofusion, and electrochemotherapy, or is undesirable, as in tumor and cardiac ablations. Understanding of how morphology affects cell viability post-electroporation may lead to improved electroporation methods. In this study, we use precisely aligned nanofiber networks within a microfluidic device to reproducibly generate elongated cells with controlled orientations to an applied electric field. We show that cell viability is significantly dependent on cell orientation, elongation, and spread. Further, these trends are dependent on the external buffer conductivity. Additionally, we see that cell survival for elongated cells is still supported by the standard pore model of electroporation. Lastly, we see that manipulating the cell orientation and shape can be leveraged for increased transfection efficiencies when compared to spherical cells. An improved understanding of cell shape and pulsation buffer conductivity may lead to improved methods for enhancing cell viability post-electroporation by engineering the cell morphology, cytoskeleton, and electroporation buffer conditions.

Experimental and theoretical model for the origin of coiling of cellular protrusions around fibers.

Protrusions at the leading-edge of a cell play an important role in sensing the extracellular cues during cellular spreading and motility. Recent studies provided indications that these protrusions wrap (coil) around the extracellular fibers. However, the physics of this coiling process, and the mechanisms that drive it, are not well understood. We present a combined theoretical and experimental study of the coiling of cellular protrusions on fibers of different geometry. Our theoretical model describes membrane protrusions that are produced by curved membrane proteins that recruit the protrusive forces of actin polymerization, and identifies the role of bending and adhesion energies in orienting the leading-edges of the protrusions along the azimuthal (coiling) direction. Our model predicts that the cell's leading-edge coils on fibers with circular cross-section (above some critical radius), but the coiling ceases for flattened fibers of highly elliptical cross-section. These predictions are verified by 3D visualization and quantitation of coiling on suspended fibers using Dual-View light-sheet microscopy (diSPIM). Overall, we provide a theoretical framework, supported by experiments, which explains the physical origin of the coiling phenomenon.

Tunneling Nanotubes between Cells Migrating in ECM Mimicking Fibrous Environments.

Tunneling nanotubes (TNTs) comprise a unique class of actin-rich nanoscale membranous protrusions. They enable long-distance intercellular communication and may play an integral role in tumor formation, progression, and drug resistance. TNTs are three-dimensional, but nearly all studies have investigated them using two-dimensional cell culture models. Here, we applied a unique 3D culture platform consisting of crosshatched and aligned fibers to fabricate synthetic suspended scaffolds that mimic the native fibrillar architecture of tumoral extracellular matrix (ECM) to characterize TNT formation and function in its native state. TNTs are upregulated in malignant mesothelioma; we used this model to analyze the biophysical properties of TNTs in this 3D setting, including cell migration in relation to TNT dynamics, rate of TNT-mediated intercellular transport of cargo, and conformation of TNT-forming cells. We found that highly migratory elongated cells on aligned fibers formed significantly longer but fewer TNTs than uniformly spread cells on crossing fibers. We developed new quantitative metrics for the classification of TNT morphologies based on shape and cytoskeletal content using confocal microscopy. In sum, our strategy for culturing cells in ECM-mimicking bioengineered scaffolds provides a new approach for accurate biophysical and biologic assessment of TNT formation and structure in native fibrous microenvironments.

Dynamic Heterochromatin States in Anisotropic Nuclei of Cells on Aligned Nanofibers.

The cancer cell nucleus deforms as it invades the interstitial spaces in tissues and the tumor microenvironment. While alteration of the chromatin structure in a deformed nucleus is expected and documented, the chromatin structure in the nuclei of cells on aligned matrices has not been elucidated. In this work we elucidate the spatiotemporal organization of heterochromatin in the elongated nuclei of cells on aligned nanofibers with stimulated emission depletion nanoscopy and fluorescence correlation spectroscopy. We show that the anisotropy of nuclei is sufficient to drive H3K9me3-heterochromatin alterations, with enhanced H3K9me3 nanocluster compaction and aggregation states that otherwise are indistinguishable from diffraction-limited microscopy. We interrogated the higher-order heterochromatin structures within major chromatin compartments in anisotropic nuclei and discovered a wider spatial dispersion of nanodomain clusters in the nucleoplasm and condensed larger nanoclusters near the periphery and pericentromeric heterochromatin. Upon examining the spatiotemporal dynamics of heterochromatin in anisotropic nuclei, we observed reduced mobility of the constitutive heterochromatin mark H3K9me3 and the associated heterochromatin protein 1 (HP1α) at the nucleoplasm and periphery regions, correlating with increased viscosity and changes in gene expression. Since heterochromatin remodeling is crucial to genome integrity, our results reveal an unconventional H3K9me3 heterochromatin distribution, providing cues to an altered chromatin state due to perturbations of the nuclei in aligned fiber configurations.

Sculpting Rupture-Free Nuclear Shapes in Fibrous Environments.

Cytoskeleton-mediated force transmission regulates nucleus morphology. How nuclei shaping occurs in fibrous in vivo environments remains poorly understood. Here suspended nanofiber networks of precisely tunable (nm-µm) diameters are used to quantify nucleus plasticity in fibrous environments mimicking the natural extracellular matrix. Contrary to the apical cap over the nucleus in cells on 2-dimensional surfaces, the cytoskeleton of cells on fibers displays a uniform actin network caging the nucleus. The role of contractility-driven caging in sculpting nuclear shapes is investigated as cells spread on aligned single fibers, doublets, and multiple fibers of varying diameters. Cell contractility increases with fiber diameter due to increased focal adhesion clustering and density of actin stress fibers, which correlates with increased mechanosensitive transcription factor Yes-associated protein (YAP) translocation to the nucleus. Unexpectedly, large- and small-diameter fiber combinations lead to teardrop-shaped nuclei due to stress fiber anisotropy across the cell. As cells spread on fibers, diameter-dependent nuclear envelope invaginations that run the nucleus's length are formed at fiber contact sites. The sharpest invaginations enriched with heterochromatin clustering and sites of DNA repair are insufficient to trigger nucleus rupture. Overall, the authors quantitate the previously unknown sculpting and adaptability of nuclei to fibrous environments with pathophysiological implications.

Quantitative biophysical metrics for rapid evaluation of ovarian cancer metastatic potential.

Ovarian cancer is routinely diagnosed long after the disease has metastasized through the fibrous submesothelium. Despite extensive research in the field linking ovarian cancer progression to increasingly poor prognosis, there are currently no validated cellular markers or hallmarks of ovarian cancer that can predict metastatic potential. To discern disease progression across a syngeneic mouse ovarian cancer progression model, here we fabricated extracellular matrix mimicking suspended fiber networks: cross-hatches of mismatch diameters for studying protrusion dynamics, aligned same diameter networks of varying interfiber spacing for studying migration, and aligned nanonets for measuring cell forces. We found that migration correlated with disease while a force-disease biphasic relationship exhibited F-actin stress fiber network dependence. However, unique to suspended fibers, coiling occurring at the tips of protrusions and not the length or breadth of protrusions displayed the strongest correlation with metastatic potential. To confirm that our findings were more broadly applicable beyond the mouse model, we repeated our studies in human ovarian cancer cell lines and found that the biophysical trends were consistent with our mouse model results. Altogether, we report complementary high throughput and high content biophysical metrics capable of identifying ovarian cancer metastatic potential on a timescale of hours.

Single Cell Forces after Electroporation.

Exogenous high-voltage pulses increase cell membrane permeability through a phenomenon known as electroporation. This process may also disrupt the cell cytoskeleton causing changes in cell contractility; however, the contractile signature of cell force after electroporation remains unknown. Here, single-cell forces post-electroporation are measured using suspended extracellular matrix-mimicking nanofibers that act as force sensors. Ten, 100 µs pulses are delivered at three voltage magnitudes (500, 1000, and 1500 V) and two directions (parallel and perpendicular to cell orientation), exposing glioblastoma cells to electric fields between 441 V cm-1 and 1366 V cm-1. Cytoskeletal-driven force loss and recovery post-electroporation involves three distinct stages. Low electric field magnitudes do not cause disruption, but higher fields nearly eliminate contractility 2-10 min post-electroporation as cells round following calcium-mediated retraction (stage 1). Following rounding, a majority of analyzed cells enter an unusual and unexpected biphasic stage (stage 2) characterized by increased contractility tens of minutes post-electroporation, followed by force relaxation. The biphasic stage is concurrent with actin disruption-driven blebbing. Finally, cells elongate and regain their pre-electroporation morphology and contractility in 1-3 h (stage 3). With increasing voltages applied perpendicular to cell orientation, we observe a significant drop in cell viability. Experiments with multiple healthy and cancerous cell lines demonstrate that contractile force is a more dynamic and sensitive metric than cell shape to electroporation. A mechanobiological understanding of cell contractility post-electroporation will deepen our understanding of the mechanisms that drive recovery and may have implications for molecular medicine, genetic engineering, and cellular biophysics.

Cell Fragment Formation, Migration, and Force Exertion on Extracellular Mimicking Fiber Nanonets.

Cell fragments devoid of the nucleus play an essential role in intercellular communication. Mostly studied on flat 2D substrates, their origins and behavior in native fibrous environments remain unknown. Here, cytoplasmic fragments' spontaneous formation and behavior in suspended extracellular matrices mimicking fiber architectures (parallel, crosshatch, and hexagonal) are described. After cleaving from the parent cell body, the fragments of diverse shapes on fibers migrate faster compared to 2D. Furthermore, while fragments in 2D are mostly circular, a higher number of rectangular and blob-like shapes are formed on fibers, and, interestingly, each shape is capable of forming protrusive structures. Absent in 2D, fibers' fragments display oscillatory migratory behavior with dramatic shape changes, sometimes remarkably sustained over long durations (>20 h). Immunostaining reveals paxillin distribution along fragment body-fiber length, while Forster Resonance Energy Transfer imaging of vinculin reveals mechanical loading of fragment adhesions comparable to whole cell adhesions. Using nanonet force microscopy, the forces exerted by fragments are estimated, and peculiarly small area fragments can exert forces similar to larger fragments in a Rho-associated kinase dependent manner. Overall, fragment dynamics on 2D substrates are insufficient to describe the mechanosensitivity of fragments to fibers, and the architecture of fiber networks can generate entirely new behaviors.

ECM in Differentiation: A Review of Matrix Structure, Composition and Mechanical Properties.

Stem cell regenerative potential owing to the capacity to self-renew as well as differentiate into other cell types is a promising avenue in regenerative medicine. Stem cell niche not only provides physical scaffolding but also possess instructional capacity as it provides a milieu of biophysical and biochemical cues. Extracellular matrix (ECM) has been identified as a major dictator of stem cell lineage, thus understanding the structure of in vivo ECM pertaining to specific tissue differentiation will aid in devising in vitro strategies to improve the differentiation efficiency. In this review, we summarize details about the native architecture, composition and mechanical properties of in vivo ECM of the early embryonic stages and the later adult stages. Native ECM from adult tissues categorized on their origin from respective germ layers are discussed while engineering techniques employed to facilitate differentiation of stem cells into particular lineages are noted. Overall, we emphasize that in vitro strategies need to integrate tissue specific ECM biophysical cues for developing accurate artificial environments for optimizing stem cell differentiation.