Substrates mimicking the blastocyst geometry suitable to revert pluripotent stem cells to naivety and method using the same

Abstract

The present disclosure refers to a substrate for use in cell culture, the substrate comprising a surface intended to be in contact with cells to be cultured, the surface comprising at least one blastocyst motif substrate (BMS) microstructure, wherein said BMS microstructure is characterized in that said BMS microstructure comprises a maximum height within the range of from 5 µm to 100 µm, a maximum width within the range of from 20 µm to 150 µm and a blastocyst scaled curvature range (BSCR) of from 15 to 62 mm-1, as well as a method of reversing primed stem cells into naïve pluripotent stem cells by use of a substrate of the invention.

Description

[0001] Substrates mimicking the blastocyst geometry suitable to revert pluripotent stem cells to naivety and method using the same

[0002] Technological Background

[0003] In nature, naive and primed states of pluripotent stem cells (PSCs) emerge sequentially in pre- and post-implantation embryos. These states are distinct in morphology, clonogenic capacity, transcriptome and epigenetic signatures. Naive pluripotent stem cells (nPSCs) have shown robust proliferation and enhanced directed differentiation, especially towards extra-embryonic lineages, as compared to their primed counterparts. Therefore, generating and stabilizing nPSCs in vitro is of great importance for gastruloid / organoid generation, modeling of early embryo, fetal anomalies, and diseases, and drug / vaccine development.

[0004] Current strategies of primed-to- naive reversion rely on small molecule or genetic approaches, which are tedious, cost intensive and subject to safety concerns. This warrants the search for robust, scalable, rapid and safe approaches.

[0005] Summary of Invention

[0006] The invention is defined by the appended claims. The description that follows is subjected to this limitation. Any disclosure lying outside the scope of said claims is only intended for illustrative as well as comparative purposes.

[0007] According to one aspect of the present invention, a substrate intended / configured for use in cell culture is provided, wherein the substrate of the invention is defined as blastocyst motif substrate (BMS).

[0008] The substrate of the invention comprises a surface intended to be in contact with cells to be cultured, the surface comprising at least one blastocyst motif substrate (BMS) microstructure, wherein said BMS microstructure is characterized in that said BMS microstructure comprises a maximum height within the range of from 5 pm to 100 pm, a maximum width within the range of from 20 pm to 150 pm and a blastocyst scaled curvature range (BSCR) of from 15 to 62 mor1, wherein the BSCR preferably defines a concave depression within the surface of the substrate. According to another aspect of the present invention, it is provided a method of culturing cells, said method comprising the use of a substrate according to the present invention.

[0009] Yet another aspect of the present invention refers to a use of a substrate according to the present invention or a method according to the present invention in culturing of stem cells; preferably in culturing stem cells to generate naive pluripotent stem cells; more preferably in culturing stem cells to reverse primed stem cells into naive pluripotent stem cells.

[0010] Further aspects of the present invention could be learned from the dependent claims and / or the following description.

[0011] In the present invention is based on the definition of specific surface topographical structures, and the analysis in situ of said complex microstructures on PSC reversion by correlating with time-resolved microscopy images. By using the substrates of the invention, it could surprisingly be shown that a specific curvature constraint in already a single direction (i.e. the blastocyst scaled curvature range (BSCR) of the substrate of the invention disclosed herein) enables the successful cell reversion through activation of cell-cell cohesion, intracellular signaling and epigenetic modulation, without necessarily recapitulating the relative radial-symmetry of blastocyst.

[0012] It is demonstrated that the reverted naivety of PSCs was sustained for at least ten days after removal of BMS. The BMS-PSCs processed higher development potential in generating embryoid bodies and teratomas, as compared to cells on non-structured substrates.

[0013] Thus, it has been shown that the BSCR disclosed herein is suitable for reverting both primed mouse and human PSCs to a more naive state.

[0014] Detailed Description of the Invention

[0015] Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings. Effects and features of the exemplary embodiments, and implementation methods thereof will be described with reference to the accompanying drawings. The present disclosure, however, may be embodied in various different forms, and should not be construed as being limited to only the illustrated embodiments herein. Rather, these embodiments are provided as examples so that this disclosure will be thorough and complete, and will fully convey the aspects and features of the present disclosure to those skilled in the art. Accordingly, processes, elements, and techniques that are not considered necessary to those having ordinary skill in the art for a complete understanding of the aspects and features of the present disclosure may not be described. In the drawings, the relative sizes of elements, layers, and regions may be exaggerated for clarity.

[0016] In the following description of embodiments of the present disclosure, the terms of a singular form may include plural forms unless the context clearly indicates otherwise.

[0017] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items. Expressions such as “at least one of,” when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

[0018] As used herein, the term "substantially," "about," and similar terms are used as terms of approximation and not as terms of degree, and are intended to account for the inherent deviations in measured or calculated values that would be recognized by those of ordinary skill in the art. Further, if the term “substantially” is used in combination with a feature that could be expressed using a numeric value, the term “substantially” denotes a range of + / - 5% of the value centered on the value.

[0019] It will be further understood that the terms “include,” “comprise,” “including,” or “comprising” specify a property, a region, a fixed number, a step, a process, an element, a component, and a combination thereof but do not exclude other properties, regions, fixed numbers, steps, processes, elements, components, and combinations thereof.

[0020] It will also be understood that when a film, a region, or an element is referred to as being "above" or "on" another film, region, or element, it can be directly on the other film, region, or element, or intervening films, regions, or elements may also be present.

[0021] Herein, the terms “upper” and “lower” are defined according to the z-axis. For example, the upper cover is positioned at the upper part of the z-axis, whereas the lower cover is positioned at the lower part thereof. In the drawings, the sizes of elements may be exaggerated for clarity. For example, in the drawings, the size or thickness of each element may be arbitrarily shown for illustrative purposes, and thus the embodiments of the present disclosure should not be construed as being limited thereto.

[0022] As used herein, the term “at least one” element refers to embodiments comprising one or more than one of said element.

[0023] In the following description of embodiments of the present disclosure, the terms of a singular form may include plural forms unless the context clearly indicates otherwise.

[0024] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and / or the present specification, and should not be interpreted in an idealized or overly formal sense, unless expressly so defined herein.

[0025] General Concept

[0026] Pluripotent stem cells (PSC) exist in two distinct states in vivo: naive and primed. Naive PSCs are derived from the blastocyst of the preimplantation embryo and can be expanded faster and more scalable without spontaneous differentiation. They exhibit broader and higher developmental potential than their primed counterparts, especially for the generation of organoids, gastruloids, and embryoids. These applications hold great promise for drug screening, disease modeling, and personalized precision medicine.

[0027] However, obtaining naive PSCs is challenging due to their transient existence in blastocysts and hard sustaining in culture vessels. Despite the long-term success of stem cell research, the in vitro primed-to-naive reversion, large-scale production, and stabilization of naive cells remain technical challenges for translational medical applications. These challenges have only been partially addressed through genetic engineering or specific degradable growth factors in combination with restricted culture conditions (e.g., using hypoxia chambers and mouse embryonic fibroblast as feeder cells). These methods are inefficient, labor-intensive, expensive, relatively non-scalable, unstable, and subject to safety concerns.

[0028] So far, there exist no medical devices specifically designed and manufactured for robust, scalable, rapid, stable, safe maintaining and stabilizing naive pluripotency for the clinical delivery and therapeutic applications.

[0029] This invention discloses means and methods to reset the naive pluripotency of primed PSCs through the utilization of culture substrate containing microscale curvatures resembling the natural habitat of naive stem cells, the blastocyst. This invention relies on a physical approach to promote the quality and application potential of pluripotent stem cells.

[0030] The provision of the substrate motifs of the present invention paves the way for medical device design and large-scale production of naive stem cells, facilitating fundamental research and applications in pharmaceutical and biomedical fields.

[0031] In nature, naive PSCs could be observed in the pre-implantation blastocyst at E3.5-4.5 for mouse and 6 days post coitum for human. Direct isolation of naive PSCs faces the processing complexity and limitation of cell number. Of note, the application on human embryos may also have ethical issues. Current strategies of the prior art to generate naive PSCs are listed below.

[0032] Genetic modification

[0033] Somatic cells were transduced / transfected with DNA or mRNA to ectopically introduce OCT4, S0X2, KLF4, cMYC genes (OSKM) in rodent system. Knockdown of Lin28a level could revert mouse primed pluripotent stem cells to their naive state. Transfection of naive associated pluripotent genes in addition to OSKM such as NANOG and KLF2, overexpression of cellular mechanosensor YAP were demonstrated to induce naive pluripotency in human somatic cells.

[0034] Disadvantages of said strategies can be summarized as follows:

[0035] So far, there is no reliable and stable genetic manipulation protocol established for reprogramming of somatic cells to naive pluripotent stem cells. The desired protein production and epigenetic modification cannot be precisely controlled by exogenous delivery of DNA and mRNA at transcription level. Therefore, the procedure is very time-consuming and may cause genomic instability, potential mutagenesis and unwanted chromosomal integration of the genes. Additionally, the reprogramming efficiency is still low.

[0036] Treatment with small-molecule chemical compounds

[0037] For mouse pluripotent stem cells, it has been documented that the naive pluripotency can be stabilized in the absence of feeder cells in vitro by supplementing soluble small-molecule chemical compounds, such as 3i medium containing MEK1 / 2, GSK3P and FGF inhibitors, and medium supplemented with leukemia inhibitory factor, MEK1 / 2 and GSK3P inhibitors or several other small molecules under a hypoxia condition. In human system, medium alone was not sufficient to stabilize the naivety. The feeder cells are indispensable. Strategies have been applied to induce and maintain the naivety by using additional panels of soluble factors such as p38 inhibitor, JNK inhibitor, aPKCi, ROCK inhibitor, and low doses of FGF2 and TGFpi with or without BRAF and SRC inhibitors.

[0038] Disadvantages of such small-molecule approaches are:

[0039] There are several limitations associated with the cocktail of small-molecule compounds used to reverse naivety. These compounds are extremely expensive, and their specificity can lead to off-target effects, especially with kinase inhibitors, which can cause cross- reactivity and side effects. Additionally, the efficiency of the reversion process is low. Furthermore, this process requires mouse-derived feeder cells and a hypoxia chamber system, which makes it unsuitable for any potential cell transplantation and translational medicine applications.

[0040] Currently, in addition to the conventional methods, there is an alternative approach that has been reported. This approach involves utilizing chitosan membranes to facilitate the selfassembly of human PSCs into 3D spheroids, allowing for long-term proliferation and maintenance of pluripotency, along with the promotion of expression of several naive- associated genes.

[0041] However, the effectiveness of naive reversion and stabilization of PSCs remains ambiguous, as the protein level of naivety markers like NANOG did not show an increase compared to the control. Additionally, chitosan may pose immunogenic risks, and factors such as batch-to-batch variation, degradation of chitosan, pH sensitivity, and by-products of ethyl (dimethylaminopropyl) carbodiimide / N-hydroxysuccinimide (EDC / NHS) could potentially introduce systemic instability and safety concerns. The spontaneous formation of 3D spheres by cells adds another layer of complexity, making it challenging to assess homogeneous functionality and systemic stability.

[0042] Herein structural parameters (width, height, and blastocyst-scaled curvature) of epiblast architecture are analysed using extensive pre-existing datasets of mouse blastocyst. Naive PSCs inhabit the concave interior of the blastocyst, characterized by trophectoderm lining with a well-defined curvature. Thus, the blastocyst-scaled curvature range (BSCR) has been defined as a quantitative descriptor considering epiblast height and width (radius: 30 pm). A BSCR value between 15 and 62 mm-1has been determined as a suitable design criterion to mimic the curvature of natural blastocyst.

[0043] We examined the naivety of induced pluripotent stem cells on the substrate areas with different well-defined micro- curvature using time-resolved, correlative in situ, and dynamic analysis, to evaluate the effect of different motifs on naivety reversion.

[0044] We found that even the motifs on substrates with BSCR in only one direction can effectively promote naivety reversion. Across uniform or randomized substrates, regardless of isotropic or anisotropic distribution, reversion efficiency is positively correlated with increased BSCR levels. While reversion efficiency within 72 hours on uniform substrates is not as high as on those with randomized microstructures, the enhancement of naive levels was significantly greater (1.5- folder). The BSCR tightly compacting cell colonies with the apical assembled actin filament and upregulated cell tension. The intracellular and intranuclear signaling cascade could be activated by structural cues, including the enhanced E-cadherin mediated cell-cell interaction, YAP activation, histone modification and gene expression. We further demonstrated that the reverted naivety of PSCs was sustained for at least ten days after removal of substrates containing BSCR. These PSCs processed the higher development potential in generating embryoid bodies and in vivo teratomas.

[0045] The approach to precisely define curvature range from nature blastocysts is novel: at the interface of epiblasts / trophectoderm in pre-implantation blastocysts, naive PSC show compacted apical cell-cell contacts due to the cellular tension of surrounding trophectoderm, which presents an axially symmetric concave geometry characterized by its height, width, and curvature. We hypothesize that a blastocyst-deduced topographical microenvironment on a cell culture substrate can revert cells to naivety by physical means. We have shown that the cell constraint, mediated by the certain curvature range within the dimensional space of the blastocyst, elicits the reversion. We collectively designate this geometrical parameter as the blastocyst scaled curvature range (BSCR).

[0046] We revealed that the curvature fitting BSCR in single direction enabled the successful cell reversion through activation of cell-cell cohesion, intracellular mechanobiological signalling and epigenetic modulation, without necessarily recapitulating the relative radial-symmetry of blastocyst.

[0047] The substrates featuring uniform surfaces with isotropic microbowls and anisotropic microgrooves within defined curvature ranges were fabricated to synchronize and maximize the effects of naivety reversion. These substrates also served as a platform to discern the relationship between BSCR counts and reversion efficiency.

[0048] This invention applied a pure physical method to revert naive pluripotency of stem cells, setting out a new direction beyond the state-of-the-art. Compared to the established strategies relying on small molecule compounds or transgene expression, reversion the naivety of PSCs via BSCR presents the advantages of being simple, efficient, robust, safe and cost-effective. This approach is highly promising for large-scale production and maintenance of naive stem cells in vitro.

[0049] The method in this invention can effectively revert PSCs to a more naive state within four days, which is around 6x faster comparing to the reported conventional chemical approaches.

[0050] This invention unravels the design criteria of substrates for triggering naivety reversion, with well-defined functional structural parameters (blastocyte scaled curvature range between 15 to 62 mm-1in at least one direction).

[0051] The reversion process in this invention involves the regulation at the epigenetic level. As a result, reverted pluripotency of stem cells can be stabilized for at least one or two weeks, which facilitates long-term storage, material transfer, cell transplantation / therapy.

[0052] We analysed the architecture of nature blastocysts (The Confocal cross views of preimplantation blastocysts (embryonic day 4.25-4.5) from literatures) and determined the dimensional limits of naive PSCs in epiblast, as well as the curvature K of the Epi / TE interface (BSCR), ranging between 15 and 62 mm-1.

[0053] We employed the substrates containing both random surface roughness and uniform microstructures (e. g. microbowl and microgrooves) with well-defined micro-curvatures to evaluate the effect of BSCR on naivety reversion. The motifs, containing a curvature value fitting the BSCR, was defined as the BSCR+ area. Time-resolved, correlative microscopy image analysis was used to position the cells on the motifs and examine their naivety marker expression, to evaluate the in-situ effect of micro-curvatures on primed-to-naive reversion of PSCs.

[0054] The motifs representing mouse blastocyst scaled curvature range efficiently promoted reversion of naivety. An increase in BSCR count positively correlated with the reversion efficiency, while the BSCR even in a single direction was sufficient to elicit the reversion. We found the apical constriction, enhanced Ecad / RAC1 signalling and YAP activation of PSCs on BSCR+ motifs, which consequently regulated histone modification (H3K27me3 and H3K4me3) of pluripotency genes. The enhancement of NANOG level in PSCs pretreated with substrates presenting BSCR persisted for at least ten days after removal from such substrates. These PSCs displayed a higher development potential in generating embryoid bodies and teratomas.

[0055] To identify the precise geometric and curvature criteria for facilitating the naivety reversion, extensive screening, and analysis of structural parameters, including width, height, and blastocyst-scaled curvature, were conducted on epiblast architecture using comprehensive pre-existing blastocyst image datasets. The determined blastocyst-scaled curvature range (BSCR) is quantitatively defined as falling between 15 and 62 mm-1, considering the dimensions of epiblast height and width (radius: 30 pm). Conducting artificial intelligence aided analysis to map, screen and precisely position the stem cells and substrate motifs with a wide range micro-curvatures including those fitting a blastocyst relevant scale.

[0056] Fabrication of substrates featuring uniform surfaces with isotropic microbowls and anisotropic microgrooves in defined curvature ranges to synchronize and maximize the effects of naivety reversion has been performed. These substrates also served as a platform to discern the relationship between BSCR counts and reversion efficiency.

[0057] Validation of reversion efficacy was accomplished through spatiotemporal analysis, correlating in situ geometric information with cellular behaviours. This involved confocal laser scanning microscopic imaging of naivety marker expression and cell occupation on different motifs of the substrate. The analysis revealed a robust correlation of BSCR motifs with naivety marker expression on substrates featuring randomized BSCR distribution. Within 24 hours of in vitro culture, 22% of mouse iPSCs were successfully reverted, with 70% located in BSCR areas. By 4 days of culture, BSCR areas achieved a reversion rate exceeding 90%. Uniform substrates with elevated BSCR levels exhibited increased reversion efficiency. Notably, in comparison to substrates with randomized microstructures, cells on uniform substrates showed a 1.5-fold higher naivety level. Reversion to naivety was evident even when the BSCR was presented in only one direction of a microstructural motif, underscoring the significance of BSCR as a highly relevant geometric factor associated with naive pluripotency.

[0058] The underlying reversion mechanism and the stability of the reverted naive PSCs were further elucidated: Microstructures within BSCR shaped iPSCs into a pyramidal shape, strengthening cytoskeleton-bound cell-cell junctions via E-cad / RAC1 , which consequently trigger YAP activation. This activation led to an increase in permissive H3K4me3 at the naive gene promoter or distal enhancer sites, along with repressive H3K27me3 at the primed gene promoters. The stability of the naive state post-reversion was confirmed by observing colony morphology, sustained levels of E-cad, YAP, and NANOG, as well as enhanced in vitro (reseeding) and in vivo (teratoma) clonogenicity.

[0059] Evaluating the naivety reversion in human PSCs, recognizing potential differences in intracellular signalling pathways compared to mouse PSCs, was conducted alongside the primary focus on mouse induced PSCs in this invention. Strikingly, BSCR areas can also efficiently induce reversion in human PSCs, leading to an enhancement of naive markers CD7 and NANOG, accompanied by a reduction in primed markers CD24, CD57, and CD90. Cells on blastocyst-motif substrates exhibited a more compact arrangement compared to those on a plain substrate, displaying a smaller cell size and robustly activated E-cadherin. Inhibiting cellcell cohesion and biomechanic sensing-associated proteins, including E-cad, RAC1 , and YAP, significantly reduced the level of the naivety marker NANOG. These findings suggest potential similarities in key components for curvature sensing and naivety reversion between mouse and human PSCs, underscoring the applicability of substrates containing blastocyst-scaled curvature for human PSC applications.

[0060] Commercialization is fundamentally possible and also attractive. Microstructured substrates for the culture of stem cells, stem cell-derived spheroids and organoids are already commercially available but not specifically designed for naive pluripotent stem cells and their derivatives such as organoids. These substrates can be used, for example, in science and diagnostics. With the new method, pluripotent stem cells can be preconditioned on substrate for one passage (3-5 days) and further transfer to traditional smooth cell culture vessels without losing their naivety within one or two weeks. Cryopreservation of reverted naive cells for a long-term storage are possible. Moreover, the new method enables the large-scale production of naive stem cells to fulfil the requirements of cell-based therapies. The new method is particularly suitable for plastic and even metal implant surface treatment to enhance their potential application for tissue engineering and regenerative medicine. There is great potential for applications in pharmaceutical research. For the development of drugs, spheroids, organoids, gastruroids, and embryoids derived from the naive stem cells obtained from the substrate of the invention can be used to investigate the effects of new drugs on individual and collective cell behaviour at tissue as well as organ level. Further, it fits the Ell “3R” scientific and technical criteria for replacing, reducing, and refining of animal studies.

[0061] According to one aspect of the present disclosure, a substrate is provided, said substrate being intended or configured for use in cell culture, the substrate comprising a surface intended to be in contact with the cells to be cultured, the surface comprising at least one blastocyst motif substrate (BMS) microstructure, wherein said BMS microstructure is characterized in that said BMS microstructure comprises a maximum height h within the range of from 5 pm to 100 pm, a maximum width w within the range of from 20 pm to 150 pm and a blastocyst scaled curvature range (BSCR) of from 15 to 62 mm-1. The substrate of the present invention is intended, suitable or configured for use in cell culture. Accordingly, the substrate of the invention or a part thereof comprises or consists of a material suitable for cell culture. Thus, at least those parts of the substrate of the invention comprising the surface intended or configured to be in contact with the cells to be cultured is formed of a material which is suitable for use in cell culture. The person skilled in the art is well aware of materials suitable for use in manufacturing of substrates intended and configured to be used in cell culture. Preferred materials comprise materials comprising or consisting of a plastic polymer, hydrogel / polymer, ceramic, or metal or metal alloys thereof, or combinations of one or more thereof. Commonly used materials comprise plastic polymers; preferably the plastic polymer / block copolymer is chosen from polystyrene (PS), polypropylene (PP), glycol-modified polyethylene terephthalate (PETG), polyethylene terephthalate (PET), polycarbonate (PC), polycaprolactone (PCL), polylactide (PLA), polydimethylsiloxane (PDMS), polyurethane (Pll), poly(p-dioxanone) (PPDO), poly(pentadecalactone) (PPDL) or mixtures thereof. More preferably the hydrogel / polymer is chosen from poly(N-isopropylacrylamide)(PNIPAM), poly(ethylene glycol) (PEG), poly(L-lactic acid-co-glycolic acid) (PLGA), poloxamers (Pluronics), polyvinyl alcohol (PVA), polyacrylamide (PAM), gelatin, collagen, laminin, fibrin, alginate, chitosan, hyaluronic acid, agarose, polydopamine or mixtures thereof. More preferably the ceramic is chosen from hydroxyapatite, tricalcium phosphate, calcium phosphate cements, bioactive glass, zirconia, alumina, silica, calcium silicate, magnesium phosphate ceramics, chitosan-ceramic composites, strontium-doped hydroxyapatite, or mixtures thereof. More preferably the metal is chosen from gold, silver, platinum, palladium, titanium and titanium alloys, stainless steel, cobalt-chromium alloys, nickel-titanium alloys, tantalum, magnesium alloys, or mixtures thereof or an alloy comprising one of the metals listed above.

[0062] The substrate of the present invention can have any form or can be part of a more complex assembly, provided the substrate is intended or configured for use in cell culture and has a surface intended to be in contact with cells to be cultured, said surface comprising at least one BMS microstructure in the sense of the present invention. The substrate may form part of, comprise or consist of a cell culture vessel or a transplant or any other object intended or configured for adherence and culturing of cells.

[0063] The substrate may be or form part of a transplant, implant or graft. In such a case, the surface comprising at least one BMS microstructure may be arranged and configured such that specific cells originating from the subject receiving the transplant migrate into / onto and populate said surface of the substrate of the invention. Alternatively or in addition, said transplant may be pre-populated with cells prior to implantation into said subject.

[0064] The substrate may be part of, comprise or consist of a cell culture vessel. The person skilled in the art is well aware of various embodiments of different cell culture vessels, wherein the exact nature and design of said cell culture vessels may depend on the type of cells to be cultured and / or on the purpose of such cell culturing. Preferably said cell culture vessel is selected from a cell culture dish, a cell culture bottle, a cell culture plate, a multi-well cell culture plate, a cell culture bucket and / or a cell culture insert suitable for insertion into one of a cell culture dish, a cell culture bottle, a cell culture plate, a well of a multi-well cell culture plate, a cell culture bucket.

[0065] Preferably, the substrate is formed of a material which can be sterilized. Sterilization can occur by use of radiation, of, gas, of disinfectants, of heat and / or other means of sterilization as well as combinations comprising one or more of said means. One way of sterilizing the substrate of the invention is by autoclaving. The substrate of the invention can be provided in non-sterile form. Preferably, the substrate of the invention is provided in sterile form, wherein at least a part of the surface comprising at least one BMS microstructure is provided in sterile form. More preferably more than 50%, more than 70%, more than 90% and / or more than 95% of the surface of the substrate of the invention comprising BMS microstructures is provided in a sterile or sterilized form.

[0066] The surface of the substrate of the invention intended / configured to be in contact with the cells to be cultured can be arranged at any position within the substrate of the invention allowing for proper culturing of cells. Preferably, said surface is arranged at the bottom of the substrate of the invention; however, it can also be positioned at other locations within the substrate depending on the cell-type to be cultured and the nature of the substrate and conditions used for cell culturing. In one example, the substrate may be or form part of a transplant or object to be placed within the human or animal body. E.g. in such a case, said surface may be provided on an outside area (e.g. an area facing away from the core of the substrate and optionally towards an environment of the substrate), or inside the porous scaffold (e.g. on the inner wall of the pores). Preferably said surface is arranged at the bottom of the substrate of the invention e.g. facing a volume, cavity or interior of said substrate or at an inner wall of the blastocyst motif substrate; more preferably said surface is arranged at the bottom or inner wall facing a volume, cavity or interior of the blastocyst motif substrate.

[0067] The substrate of the invention comprises a surface intended / configured to be in direct or indirect contact with the cells to be cultured, said substrate comprising at least one BMS microstructure. The at least one BMS microstructure is designed to mimic the microenvironment of the pocket housing naive PSCs in the blastocyst and, thus, is preferably formed to be concave, i.e. to have a cross-section with a concave profile.

[0068] The at least one BMS microstructure of the substrate of the invention exhibits a maximum height h (i.e. depth in case the BMS microstructure being concave) within the range of from 5 pm to 100 pm. The maximum height h of the at least one BMS microstructure is expressed as a positive height and is determined between the midpoint of the width line representing the diameter, connecting opposing points of the rim of the BMS microstructure (i.e. opposing points lying on the interface, where the curvature of the BMS microstructure meets the plain surface of the substrate surrounding the BMS microstructure), and the point at the bottom of the curvature of the BMS microstructure opposing the midpoint in a direction perpendicular to the width line. The maximum height h of the BMS microstructure can be determined using micro computed tomography (micro-CT) as described in more detail in the experimental part of the specification (see also Figure 1a, Fig. 1 b-c, Fig. 17e and Table 1-3).

[0069] Preferably, the at least one BMS microstructure comprises a maximum height h within the within the range of from 5 pm to 48 pm; preferably of from 5 pm to 48 pm, more preferably of from 5 pm to 40 pm, even more preferably of from 8 pm to 38 pm; or a range selected from 10 pm to 35 pm or 12 pm to 30 pm.

[0070] The at least one BMS microstructure of the substrate of the invention exhibits a maximum width w within the range of from 20 pm to 150 pm. The maximum width w of the at least one BMS microstructure is expressed as a positive width and is determined by the diameter through the centre point of the BMS microstructure, connecting opposing points of the rim of the BMS microstructure (i.e. opposing points lying on the interface, where the curvature of the BMS microstructure meets the plain surface of the substrate surrounding the BMS microstructure). The maximum width w of the BMS microstructure can be determined using micro computed tomography (micro-CT) as described in more detail in the experimental part of the specification (see also Figure 1a, Fig. 1 b-c, Fig. 17d and Table 1-3).

[0071] Preferably, the at least one BMS microstructure comprises a maximum width w within the range of from 25 pm to 80 pm; preferably of from 29 pm to 79 pm; more preferably 30 pm to 75 pm, even more preferably of from 40 pm to 72 pm; or a range selected from 45 pm to 70 pm or 55 pm to 65 pm. The at least one BMS microstructure of the substrate of the invention exhibits a blastocyst scaled curvature range (BSCR) of from 15 to 62 mnr1(see also Fig. 1a and Fig. 17c). The blastocyst scaled curvature, K, of a given BMS microstructure is calculated as defined in more detail in the experimental section of the specification. Based on voxel datasets from micro computed tomography (micro-CT), the spatial information of the BMS surface was calculated and mapped. A scaled three-point approach (see Fig. 1b,c and Fig. 19) was developed to measure the asymmetrical curvatures in 18 discrete directions around each surface point on the substrate. A plane cut was made to measure the curvature of point A on the defined direction. Two other points, C and E, are chosen, equidistant from A in the given plane, which defines a circle with radius r in the given plane cut. The total analyzed length for the measured points is 2L = 60 pm. The scaled three-point approach for curvature, K, measurement as used in this invention is based on Heron’s formula. On a surface S for any given point p, curvature is analyzed around the point itself 360 degrees (see Fig. 19). This is done by giving a direction in the form of a vector, v, then two other points are selected by going in the positive direction of the vector and the negative direction of the vector. Once the three points are selected, a system of equations can be written to solve for a circle that touches all three points. The inverse of the radius is the curvature for that point p in the direction v. This is then repeated for n directions for each point. The curvature, K, is computed at each 10-degree increment, yielding 36 curvatures. However, due to symmetry only 18 are unique and, thus are sufficient for determining the curvature K. The magnitude of vector, v, is the scale, with which the curvature K was analyzed. The magnitude for v was set to 30 pm, as this reflected half the width of an Epi (naive cell cluster) which is ~60 pm (see Fig. 19).

[0072] From a cross-sectional view, the three points are chosen by selecting the given point which is where curvature is to be calculated, p2 = (x2,y2), and then moving a certain distance negatively and positively, in each direction along the curve to obtain p1 = (x1 ,y1) and p3 = (x3,y3). From these three points, there is only one solution for fitting a circle on all three points, which can be explained from the equations outlined below:

[0073] The equation of the circle is described by the equation:

[0074] Ax2+ Ay2+ Bx + Cy + D = 0

[0075] And substituting the three points which does lie on the circle, a system of equations is obtained which can be described by the determinant.

[0076] And the coefficients A,B,C, and D can be found by solving the following determinants

[0077] The center point (x,y) can then be found:

[0078] B C x = - , y = -

[0079] 2Ay2A

[0080] And from this the radius can be calculated:

[0081] And curvature is then:

[0082] Preferably, the at least one BMS microstructure of the substrate of the invention comprises a BSCR within the range of from 16 to 45 mm-1, preferably of from 18 to 42 mm-1; more preferably of from 20 to 40 mm-1; even more preferably of from 25 to 35 mm-1.

[0083] The diameter of a single PSC is approx. 15-20 pm. The blastocyst motif (BM) of the substrate of the invention applied for a PSC cell cluster should be larger than a single cell occupied area. Preferably, BM comprise a culture area larger than 300 pm2, preferably larger than 500 pm2. It has surprisingly been found that, in order to be suitable to revert primed PSCs to naivety, it is not necessary that the BMS microstructure exhibits the blastocyst scaled curvature within the claimed BSCR in all or the majority of directions. In fact, it turned out that the claimed effect was already achieved when the BMS microstructure shows said curvature within the claimed BSCR in at least one direction. This has the effect that the BMS microstructure is not limited to bowl-like structures but can also be realized with groove-like structures, wherein the curvature within the claimed BSCR is e.g. realized perpendicular to the longitudinal axis of the groove. Further, the at least one BMS microstructure can be radial symmetric or lateral symmetric; preferably the at least one BMS microstructure has the shape of a bowl or a groove.

[0084] The substrate of the invention comprises a surface having at least one BMS microstructure. However, the substrate is not limited to the presence of only a single BMS microstructure. The substrate of the invention preferably has a surface which comprises a plurality of BMS microstructures, wherein the plurality of BMS microstructures can be composed of BMS microstructures of the same or different shape and orientation. Said plurality of BMS microstructures are arranged on said surface in an unregular geometric pattern, an orderly geometric pattern or in a mixture of both. For example, the plurality of BMS microstructures can be present in the form of bowls or in the form of grooves or in a mixture of bowls and grooves, wherein said bowls and grooves may be present in an ordered manner e.g. arranged in rows and / or columns, wherein the columns are preferably present in the same orientation in parallel to each other. Alternatively, said bowls and columns may be arranged on the surface of the substrate of the invention in an unordered, randomized pattern.

[0085] In a preferred embodiment, the BMS microstructures (e.g. the bowls and / or columns / grooves) are arranged on the surface of the substrate of the invention in a predetermined density 1-273 bowls per mm2, and 1-260 grooves per mm2, wherein the distance from each of the BMS microstructure to its neighbour may be different or the same and is preferably in the range of from 0 to 60 pm, more preferably of from 5 pm to 55 pm.

[0086] The substrate of the invention is intended or configured for use in cell culture, preferably in culture of cells to be cultured under adherent conditions. In order to allow for proper adhesion of cells to be cultured in or on the substrate of the invention. The surface intended to be contacted with the cells to be cultured or the at least one BMS microstructure arranged thereon can be coated in order to promote or allow cell adhesion to said surface or BMS microstructure. The person skilled in the art is well aware of methods and means for coating of cell culture substrates based on the cell-type to be cultured. Preferably said surface and / or at least one BMS microstructure is coated with at least one polymer or extracellular matrix protein / component; more preferably the polymer is chosen from poly(ethylene glycol) (PEG), polyvinyl alcohol (PVA), poly-L-lysine (PLL), poly-D-lysine (PDL), polydopamine, chitosan, silk fibroin, and any mixture thereof, and / or the extracellular matrix protein / component is chosen from laminin, collagen, gelatine, entactin, hyaluronic acid, heparin sulphate proteoglycans, Matrigel / Geltrex, and any mixture thereof

[0087] The present invention is also directed to a method of culturing cell, wherein said method is characterized in that a substrate of the invention is used for culturing of cells. The person skilled in the art is well aware of various methods of culturing cells, wherein the exact details of such cell culturing methods depend on the nature of the cells to be cultured. Thus, selection of cell culture medium, atmosphere, temperature, time period of cell culture and other cell culture conditions will depend on the type of cells to be cultured and selection of which are within the knowledge, skill and ability of the skilled person or can be determined without undue burden.

[0088] The method of the invention preferably comprises the step of bringing cells to be cultured into contact with the substrate of the invention so that at least some of the cells are in direct or indirect contact with the at least one BMS microstructure. This step may comprise seeding of the cells on said surface of the substrate of the invention so that the cells directly or indirectly adhere to said surface. In a second step of the method of the invention, the cells are cultured; preferably the cells are cultured on the substrate for at least 3 hours; more preferably for at least 12 hours; even more preferably for at least 24h.

[0089] Pluripotent stem cells (PSCs) are a particularly potent type of stem cells that can maintain an undifferentiated state indefinitely and can differentiate into all cell types from three germ cell layers of the embryo. PSC exists in two distinct states in vivo: naive and primed. The specific naive markers include NANOG, STELLA, SSEA1 for mouse, and CD7, NANOG, E-cad for human. The specific primed markers include ZIC2 for mouse and CD24, CD57, CD90 for human. Naive PSCs are derived from the blastocyst of the preimplantation embryo and can be expanded faster and more scalable without spontaneous differentiation. They exhibit broader and higher developmental potential than their primed counterparts. The present invention is also directed to a method of generating naive PSCs, of expanding naive PSCs or reverting PSCs to naivety, wherein said method is characterized in that a substrate of the present invention is used for culture of said cells.

[0090] Further, the present invention is directed to the use of a substrate of the invention or a method of the invention in culturing of stem cells; preferably in culturing stem cells to generate naive PSCs; more preferably in culturing stem cells to reverse primed stem cells into naive PSCs.

[0091] The present invention is further exemplified by the experiments and data provided below.

[0092] Brief Description of the Drawings

[0093] Fig. 1. Design and motif analysis of blastocyst-motif substrate (BMS). a, Schematic representation of a mouse blastocyst (pre-implantation; E4.25-E4.5). The outer layer of nPSCs experiences curvatures in the blastocyst-scaled curvature range (BSCR, yellow curve), measured from the Epi / TE interface, b-d, Computational analysis of BMS surface topographical features, b, Scaled three-point approach for evaluating curvature. The curvature K was analyzed in 18 directions for each point using a scale length (CE) of 60 pm similar to the width of the Epi. c, A representative example model showing the curvature value of the given point A in 18 directions, d, Left: each point on the topographical surface was allocated a motif according to the mean curvature (K): Convex ((K) < -2.5 mm-1, blue), Flat (-2.5 mm'1< (K) < 2.5 mm-1, gray), Concave «K) > 2.5 mm-1, red). Middle: the BSCR+ area (yellow) included points where K was within the BSCR in at least one direction. Right: Proportion of the points with different BSCR counts on BMS, correlated with of Convex, Flat and Concave motifs, e, Schematic illustration of overlaid topographical maps of BMS with time-resolved cell images to evaluate the reversion and expansion of PSCs.

[0094] Fig. 2. BMS boosts the naivety of PSCs through BSCR motif, a, The levels of naive pluripotent markers NANOG, STELLA, OCT4, SSEA1 , and primed marker ZIC2 in day- 5 PSCs on different substrates. Positive control (TCP, 2i / L) for naive state was set as 1 (n=3 independent experiments. * p < 0.05, ** p < 0.01 , *** p < 0.001 , one-way ANOVA with Bonferroni’s multiple comparisons test), b, Representative BMS topographical map and PSC image. Cells were stained at day 3 with Hoechst 33342 to visualize the nuclei of whole population (blue), while the NANOGhi(green) and STELLAhi(white) cells were filtered out to show the naive population, i) bright field (BF) image of BMS and nuclei staining of PSCs; ii) Naive PSCs overlaid with BMS motif map: Concave (red), Convex (cyan), Flat (gray), BSCR+ (yellow) and BSCR- (black), iii) Naive PSC distribution in BSCR+ and BSCR- areas in Concave region. Scale bars, 100 pm. c-f, Percentage of nPSCs (NANOGhiand STELLAhi) out of total cells on indicated motifs was analyzed over time to elucidate the effect of BSCR (c, d) and BSCR counts (e, f) on PSC naivety. BSCR-low, -med and -hi represent the BSCR count of 1-6, 7-12 and 13-18 respectively. (n=4 independent experiments, two-way ANOVA with Bonferroni’s multiple comparisons test, for effects of BSCR and time, *p < 0.05, **p < 0.01 , ***p < 0.001 , ****p < 0.0001).

[0095] Fig. 3. BSCR elicits in situ naivety reversion of PSCs. a-d, Early-stage cell-BMS interaction was examined 9 hours after cell seeding. Representative bright field, nuclei and NANOG staining images (a) and quantitative analysis of cell distribution (b), mean fluorescence intensity (MFI) of NANOG (c) and size (d) of PSCs, located inside and outside BSCR areas. Scale bars, 100 pm. (Cell density and NANOG: n=4; Cell size: nceii=220 and 228 for BSCR- and BSCR+ groups; N.S., non-significant, Student’s t test). e, Dynamics of NANOG-GFP expression of PSCs. Cells cultured on TCP with 1 i and 2i / L media were used as negative and positive control of naivety. Scale bar, 100 pm. f, Migration of PSCs (left: whole polulation; right: NANOG-GFPhinPSCs) inside BSCR+ area and crossing over the BSCR border on BMS in the indicated time frames. Images were recorded within 60 min (left panel) and 90 min (right panel) intervals. Scale bars, 100 pm. (nceii=93 and nceii=31 for d0-d1 and d1-d3 cell tracking, **p < 0.01, one-way ANOVA with Bonferroni’s multiple comparisons test). The outlines of BSCR+ areas were illustrated with yellow dash lines.

[0096] Fig. 4. BMS reverts PSCs to naive state via E-cad / RAC1 signaling, a-b, Representative immunofluorescence images of F-actin (red) and E-cad (orange) in PSC colonies on BMS: top-view (x-y plane) and side-view (x-z plane, optical cross section from top-view along white dot lines). Cell nuclei were stained with DAPI. Scale bar 50 pm. c-e, Quantification of E-cad expression using flow cytometry, active RAC1 and FAK activity (Y397 phosphorylated FAK (pFAK) / total FAK (tFAK) ratio) with ELISA, for PSCs on Plain and BMS (n=3 independent experiments; ** p < 0.01 , **** p < 0.0001 , Student’s t test), f, The level of NANOG, STELLA and ZIC2 in day-5 PSCs on BMSs with and without E-cad, RAC1 and FAK inhibition. The value of untreated PSCs was set as 1 (n=3 independent experiments; *** p < 0.001 , **** p < 0.0001 , one-way ANOVA with Bonferroni’s multiple comparisons), g, Dynamic changes of NANOG-GFP in PSCs growing on BMS for 5 days with and without E-cad and RAC1 inhibition. Scale bar, 100 pm. h, Fraction of nPSCs (GFPhi) out of total GFP+ cells on different motifs over time, with and without inhibitor treatment. The result was expressed as ratio of areas covered by GFPhicells and GFP+ cells, i, MFI of GFP in BSCR+ and BSCR- areas over time (n=3 independent experiments; ****p < 0.0001 , two-way ANOVA with Bonferroni’s multiple comparisons test, for effects of BSCR and time), j, Representative time-lapse images showing the inhibition of E-cad / RAC1 signaling abolished the naivety of PSCs inside BSCR+ area. Scale bar, 100 pm. For g and j, the BSCR+ areas were circled with yellow dash lines.

[0097] Fig. 5. Epigenetic reversion of PSCs to naive state on BMS mediated by YAP and histone modification, a, Flow cytometry analysis of total YAP (tYAP), S127 phosphorylated YAP (pYAP), and pYAP / tYAP ratio in PSCs from Plain and BMS, with and without RAC1 inhibition (n=3 independent experiments; * p < 0.05, ** p < 0.01 , *** p < 0.001 , Student’s t test for comparing Plain- vs. BMS-PSCs or untreated vs. inhibition groups), b, The level of NANOG, STELLA and ZIC2 in day-5 PSCs on BMS with and without YAP inhibition. The level of untreated group was set as 1 (n=3 independent experiments; ** p < 0.01 , *** p < 0.001 , Student’s t test). ChlP-PCR analysis of H3K27me3 (c) and H3K4me3 (d) levels at promoter regions of Nanog, Zic2 and at distal enhancer region of Oct4 in the absence and presence of YAP inhibitor. Data were normalized by total H3 value in each group, respectively. Positive control (TCP, 2iL) group was set as 1 (n=3 independent experiments; * p < 0.05, ** p < 0.01 , *** p < 0.001 , **** p < 0.0001 , one-way ANOVA with Bonferroni’s multiple comparisons test for groups without YAP inhibition;#p < 0.05,##p < 0.01 ,###p < 0.001 , inhibition vs. corresponding untreated group, and55p < 0.01 ,5555p < 0.0001 , Student’s t test), e, Proposed mechanism for nativity reversion at the epigenetic level. Left: Side view of the mouse PSC nuclei in BSCR+ area (yellow curve). Right: Scheme of intracellular and intranuclear signaling events triggered by BSCR.

[0098] Fig. 6. BMS stabilizes naive pluripotency of PSCs. a, Scheme for analyzing naive pluripotency stability of PSCs pre-conditioned from substrates. PSCs were preconditioned on Plain and BMS for 5 days, followed by re-seeding on laminin coated TCP for monolayer culture, on uncoated TCP for EB formation, and by injection for teratoma generation, b, Representative images showing the dynamic change of YAP and E-cad level in PSCs reseeded on laminin coated TCP. Quantitative analysis of E- cad levels (c), pYAP, tYAP levels, pYAP / tYAP ratio (d) and NANOG levels (e) in Plain- Re and BMS-Re PSCs (n=3 independent experiments; * p < 0.05, ** p < 0.01 , *** p < 0.001 , **** p < 0.0001 , two-way ANOVA with Bonferroni’s multiple comparisons). Size (f), YAP staining (g) and YAP phosphorylation level (h) of EBs, which were formed from reseeded PSCs in uncoated 96-well flat TCP. EBs grew for 2 days in f and 3 days in g, h. (f: mean ± SEM, n=100 EBs from 3 independent experiments; **** p < 0.0001 , Student’s t test; g: scale bar, 100 pm; h: n=6 independent experiments; * p < 0.05, Student’s t test. The value of Plain-Re group was set as 1.). i. YAP staining of EBs, which were formed by PSCs derived from BMS and Plain substrates in a V-bottom culture plate for 5 days to achieve the similar EB diameter. Scale bar, 100 pm. j. Representative images of teratomas formed in mice by injection of pre-conditioned PSCs. k, Representative histological images and volume of teratomas derived from injected PSCs. (Scale bar, 250 pm. Mean ± SEM, n=3 independent experiments; ** p < 0.01 , two-way ANOVA with Bonferroni’s multiple comparisons test).

[0099] Fig. 7. Enhanced naivety of PSCs on BMS. a. Real-time PCR array analysis of naive and primed pluripotent gene expression in PSCs on different substrates, b. Representative fluorescence microscopic image of SSEA1 (green) and nuclei (blue) of PSCs on Plain and BMS substrate, c. Quantitative analysis of the percentage of SSEA1+ / SSEA4- cell population in day-5 PSCs on different substrates (n=6 independent experiments; **** p < 0.0001 , Student’s t test)., d. Heat map of real-time PCR array screening of germ layer differentiation associated genes in day-5 PSCs. Percentage of NANOGhi(e) and STELLAhi(f) PSC covered area compared to the whole area of corresponding motifs (n=4; ** p < 0.01 , two-way ANOVA with Bonferroni’s multiple comparisons test), g. Analysis of intensity of NANOG in cells located in BSCR+ areas with different counts, h. Comparison of NANOG intensity of PSCs located in Concave+ and Concave- motifs with one out of 18 directions fitting BSCR (BSCR 1) and without BSCR (BSCR 0). i. Quantification of the proportion of day-3 NANOGhicells in Concave+ areas and BSCR+ motifs (n=3; * p < 0.05, Student’s t test).

[0100] Fig. 8. Primed-to-naive reversion of PSCs on BMS. Representative images and quantitative analysis of PSC necrosis (a) and apoptosis (b) in BSCR+ and BSCR- areas at day 0 (9 hours post cell seeding) and day 3 (n > 8 images for necrosis and n > 5 images for apoptosis; N.S. statistically non-significant; * p < 0.05, Student’s t test), c. Detection of NANOG-GFPhinPSCs on BSCR+ area of BMS from 12 h to 39 h post cell seeding, d. Expansion of NANOG-GFPhinPSCs from BSCR+ to BSCR- areas. The outlines of BSCR+ areas were illustrated with yellow dash lines. Scale bar, 100 pm.

[0101] Fig. 9. PSC naivety and proliferation activity on different substrates. Representative fluorescence images of Ki67+ proliferating cells, NANOG-GFPhinaive PSCs in BSCR+ and BSCR- areas of BMS, and on Plain substrate. Scale bar, 100 pm. The outlines of BSCR+ areas were illustrated with yellow dash lines. 1i-PSCs, Mixed PSCs (Naive: Primed = 1 :4 in number) were maintained under primed PSC culture condition in 1i medium. 2i / L-PSCs used as positive control were cultured under naive PSC culture condition in 2i / L medium.

[0102] Fig. 10. Quantification of PSC naivety and proliferation activity on different substrates. The NANOG-GFPhiproportion, cell density and percentage of Ki67+ proliferating cells in BSCR+, BSCR- areas of BMS and on Plain substrate within 48 h post cell seeding were quantified based on the fluorescence images. For each group, at least 5 randomly selected image fields were analyzed (nceiis > 646 for each group; * p < 0.05, ** p < 0.01 , **** p < 0.0001 for effect of microstructures, two-way ANOVA; BSCR+ vs. Plain: § p< 0 05, §§§§ p< 0.0001 ; BSCR+ vs. BSCR-: ## p< 0.01 , #### p< 0.0001; Bonferroni’s multiple comparisons test).

[0103] Fig. 11. Topography of microbowl and microgroove substrates, a. Height map reconstruction of substrates based on optical profilometry analysis, b. Representative scanning electron microscopic images of microstructured substrates. Confocal microscopic images of tilted-view (c) and side-view (d) of laminin-coated microbowls and microgrooves. Scale bar, 200 pm.

[0104] Fig. 12. Primed-to-naive reversion and expansion of PSCs on substrates with uniform motifs, a. Representative confocal microscopic images of NANOG-GFP PSCs on microbowls and microgrooves with different BSCR counts at day 3 after cell seeding. Scale bar, 100 pm. b. Quantification of the area fraction of GFPhiin GFP+ cells and MFI of GFP in PSCs growing on microbowls and microgrooves with different BSCR counts for 3 days (n=5 independent experiments; ****p < 0.0001, two-way ANOVA with Bonferroni’s multiple comparisons test, for effects of BSCR and time), c. the area fraction of GFPhiin GFP+ cells and MFI of GFP in day-3 PSCs on BSCR+ microbowls with and without E-cad, RAC1 and YAP inhibition (n=3 independent experiments; **** p < 0.0001 , one-way ANOVA with Bonferroni’s multiple comparisons), d. Quantification of fraction and MFI of SSEA1+ PSCs on BSCR+ and BSCR- microbowls (n=5 independent experiments; * p < 0.05, **** p < 0.0001 , one-way ANOVA with Bonferroni’s multiple comparisons), e. Representative confocal images showing the alteration of TBX3 and STELLA in PSCs on microbowls with different BSCR counts. Scale bar, 50 pm. Cell cycle progression patterns of day-1 (f) and day-3 (g) PSCs on microbowls with different BSCR counts (n=3 independent experiments; * p < 0.05, ** p < 0.01 , *** p < 0.001 , **** p < 0.0001 , two-way ANOVA with Bonferroni’s multiple comparisons test), h. Expansion curves of PSCs on microbowls with different BSCR counts over time (n=3 independent experiments; **** p < 0.0001 , two-way ANOVA with Bonferroni’s multiple comparisons test, for effects of BSCR and time).

[0105] Fig. 13. PSC compaction on BMS. a. Representative top- and side-view of confocal microscopic images and quantitative analysis showing spatial organization of PSC colonies in crater and channel like areas on BMS. Cell nuclei were stained with DAPI (n>9; ** p < 0.01 , **** p < 0.0001 , one-way ANOVA with Bonferroni’s multiple comparisons test), b. Imaging analysis of cell density, top-view colony area, and flow cytometry analysis of relative cell size (Cell density: n=10, ncolony=15 and 20 for Plain and BMS substrate; Colony area: n=8 and 22, ncolony=53 and 91 for Plain and BMS substrate, respectively; Cell size: n=3; N.S., non-significant, ** p < 0.01 , **** p < 0.0001 , Student’s t test), c. The flow cytometry analysis of NANOG of day-5 PSCs on Plain substrates with and without high cell seeding density (5x104 / cm2). TCP, 2i / L control was set as 1 (n=3; N.S., non-significant; ** p < 0.01 , one-way ANOVA with Bonferroni’s multiple comparisons test). Representative confocal images and (d) the quantification of pMLC2 level (e) in PSC colonies at Concave+ and Concave- areas (n=3; **** p < 0.0001 , Student’s t test), f. Comparison of pMLC2 levels of cells using flow cytometry (n=3 independent experiments; * p < 0.05, Student’s t test), g. Contractility assay based on the PSCs / collagen gel size measurement. The cells were harvested from Plain and BMS with / without E-cad inhibition (n=3; * p < 0.05, ** p < 0.01 , Student’s t test).

[0106] Fig. 14. Primed-to-naive reversion of human PSCs on BMS. a. Representative bright field (BF) image and laser scanning microscopic images of human PSCs on BMS at day 5. The nuclei (blue) were stained with Hoechst 33342 to visualize the whole population, and the NANOGhi(green) and STELLAhi(red) cells were filtered out to show the naive population. Scale bars, 100 pm. b. Flow cytometry analysis of naive markers (CD7, NANOG) and primed markers (CD24, CD57, CD90) of day-8 human PSCs adapted in RSeT medium on TCP (Pos Ctrl), on Plain and BMS substrates in IPS-brew medium (n=3; * p < 0.05, ** p < 0.01 , *** p < 0.001 , **** p < 0.0001 , one-way ANOVA with Bonferroni’s multiple comparisons test), c. Flow cytometry analysis of relative cell size (based on FSC-A value) of human PSCs cultured on Plain and BMS surfaces (n=6, **** p < 0.0001, Student’s t test), d. Quantitative analysis of E-cad+ fractions and E-cad expression levels of cells under different culture conditions (n=3; * p < 0.05, ** p < 0.01 , *** p < 0.001 , one-way ANOVA with Bonferroni’s multiple comparisons test), e. The level of NANOG in day-8 human PSCs on BMS with and without E-cad, RAC1 and FAK inhibition. The level of untreated PSCs was set as 1 (n=3 independent experiments; *** p < 0.001 , **** p < 0.0001 , one-way ANOVA with Bonferroni’s multiple comparisons).

[0107] Fig. 15. YAP activation and cell proliferation in response to culture on a BMS substrate, a. Real-time PCR array analysis of YAP signaling suppressor and target genes in day- 3 PSCs from Plain and BMS substrates, b. The representative flow cytometry dot plots showing the phosphorylated level of AmotLI (S262) in PSCs in the indicated conditions. c. Percentage of cells in the phases of cell cycle on Plain and BMS substrates (n=9; ** p < 0.01 , **** p < 0.0001 , two-way ANOVA with Bonferroni’s multiple comparisons test). d. Growth curve of PSCs on Plain and BMS substrates, with and without RAC1 inhibition (n=9; N.S, non-significant; **** p < 0.0001 , two-way ANOVA with Bonferroni’s multiple comparisons test), e. Alkaline phosphatase staining of PSCs on Plain and BMS at indicated time points. Scale bar, 5 mm.

[0108] Fig. 16. BMS stabilizes naive pluripotency of PSCs. a. Morphology of reseeded PSCs preconditioned on different substrates. Scale bar, 50 pm. b. Proliferation curves of reseeded PSCs. n=4 independent experiments; **** p < 0.0001 for main effect of substrate (two-way ANOVA). Western blot analysis of E-cad (c), pYAP (at S127) and tYAP levels (d) in day-3 EBs formed by reseeding PSCs pre-conditioned on Plain (Plain- Re) and BMS (BMS-Re) substrates. GAPDH was used as a loading control, e. Cell cycle progression patterns of day-3 EBs (n=3 independent experiments; ** p < 0.01 , *** p < 0.001 , two-way ANOVA with Bonferroni’s multiple comparisons test). Fig. 17. Characterization of pre-implantation blastocysts.

[0109] Schematic illustration of the methodology (a) and the representative confocal image and formulas (b) for analyzing the curvature of outer layer of nPSCs in blastocyst, (b) blastocyst image from4; permission will be obtained. Scale bar, 20 pm. The scatter plots showing the curvature distribution (c), width (d), height (e) and density (f) of outer layer of nPSCs at Epi / TE interface in blastocysts (n = 36).

[0110] Fig. 18. Motif and curvature distribution of BMS surfaces. a. Reconstructed 3D image of polystyrene BMS1 based on micro-CT. b. Height profiles of BMS1 - 4. The total diameter is 10.8 mm. c. Examples of radar chart curvature plots of BMS3 using the “scaled three-point curvature” with a scale of 30 pm. The left column shows the location on the BMS3, the middle column shown a zoomed in image of the region of interest, and the right column shows the radar chart curvature, K, plots. Here the values between BSCR 15 mm-1< K < 62 mm-1are colored yellow, and the black circle marks zero curvature, d. Motif maps of BMS1 - BMS4. (K) Convex ((K) <-2.5 mm’ 1, blue), Flat (-2.5 mm’1< (K) < 2.5 mm-1, gray), Concave «K) > 2.5 mm-1, red). For the Concave motif regions we focus on the subset BSCR (15 mm’1< K < 62 mm-1, yellow), e. Distribution of average curvature (K) for the polymeric microstructured BMS1-BMS4.

[0111] Fig. 19. Scaled three-point approach for curvature measurement.

[0112] Schematic illustration of scaled three-point approach for curvature measurement, a. Schematic representation of an arbitrary surface, S, in R3 space. Curve, C, lies on S and point p lies on S and within C. The normal vector, N, is perpendicular to C at p and the tangent vector, t, is tangent to C at p. The curvature vector, k, pointing in the direction of the curvature unit normal vector, n, and is composed of the normal curvature vector, kn, and geodesic curvature vector, kg. b. Schematic showing the vector-valued function r which is a function of the Lagrangian surface coordinates u and v, pointing from the origin to point p on curve C within surface S. c. 3D representation of the method. A point A on a surface is chosen around which several plane cuts (green squares) are made by rotating around A in the z-axis. For each plane cut two other points, C and E, are chosen, equidistant from A in a given plane (red square). This defines a circle with radius r in the given plane cut. Illustrative 3D example of the points chosen (d) and 2D view of the plane (e) on the surface, the total analyzed length for a given curvature value is 2L = 60 pm. f. Example 3D surface defined by the equation z=1.2*sin(0.5*x)+1.2*cos(0.5*y). g. Calculated motif color map: BSCR > 0 (yellow), Concave (red), Flat (grey), Convex (blue), (i) Motifs without BSCR shown, (ii) Motifs with BSCR shown.

[0113] Fig. 20. Correlative analysis integrating geometrical and biological information. a. Co-localization of images from laser scanning confocal microscope and micro-CT. b. Motif masks and LSM fluorescence staining images for in situ analysis, c. Overlay images of NANOGhiand STELLAhiwith different motif images. Scale bar, 500 pm.

[0114] Fig. 21. Characterization of PSCs with naive and primed-like states. a. Scheme of cell manipulation and gating strategy for flow cytometric analysis of MACs- based PSC purification, b. Morphology of naive (in 2i / L medium) and primed-like (in 1i medium) PSCs. Scale bar, 100 pm. Flow cytometry analysis of NANOG reporter-GFP (c) and quantification of NANOG protein level (d) TCP,2i / L group was set as 1 . (n=3; *p < 0.05, Student’s t test).

[0115] Fig. 22. Characterization of laminin (LMN) coated Plain and BMS substrates. a. Confocal microscopic images of top-view (i) and side-view (ii) of LMN coated Plain and BMS substrates. Scale bar, 50 pm. b. Quantification of LMN density in different surfaces based on the analysis on confocal images using Imaged software (a.u.: arbitrary units). (npiain=20 images, nConcave / convex=10 images, two-way ANOVA with Bonferroni’s multiple comparisons test), c. Quantification of the LMN adsorption on Plain and BMS via ELISA (n=3, one-way ANOVA with Bonferroni’s multiple comparisons test), d. The surface wettability of Plain and BMS substrate with and without LMN determined via contact angle measurement (n=10 measurements of 5 locations of each sample. **** p < 0.0001 , two-way ANOVA with Bonferroni’s multiple comparisons test).

[0116] Fig. 23. Threshold determination for the confocal images of BMS-PSCs.

[0117] The mean fluorescence intensity (MFI) of NANOG and STELLA of cells growing on the Plain surface was analyzed and used as a threshold to distinguish the different levels of fluorescence signals on BMS substrate. The fluoresce signals of NANOG and STELLA lower than the threshold were removed and the remained fluorescence was defined as “NANOG High (NANOGhi)” and “STELLA High (STELLAhi)”. Scale bar, 300 pm.

[0118] Fig. 24. Image processing and analysis for naivety reversion and cell proliferation. The flowchart showed an example of analysis of Ki67 expression, GFP level and nuclei distribution of cells on BSCR+ area of a BMS substrate.

[0119] Experimental section

[0120] Here we demonstrate that a blastocyst-deduced topographical microenvironment on a cell culture substrate can revert cells to naivety by physical means. It appears that the cell constraint, mediated by the certain curvature range within the dimensional space of the blastocyst, could elicit the reversion. We collectively designate this parameter as the blastocyst scaled curvature range (BSCR). When the micro-topographical units on a substrate encompass BSCR, they are referred to as BSCR+ motifs. We intend to address the question, whether the reversion requires for BSCR in a radial-symmetric manner, as provided by the Epi / TE interface, or in only a limited number of directions. However, given the impracticality of side-by-side integration of a multitude of BSCR motifs in a closed 3D architecture like blastocyst, a structured, i.e. , open 2.5D polymeric substrate should be engaged.

[0121] To test our approach, the blastocyst motif substrate (BMS) was designed and fabricated featuring thousands of randomly varied motifs, covering not only biological size and shape heterogeneity but also following our reductionist paradigm. Electrical discharge machining (EDM) was utilized to create the microstructure on metal mold surfaces. The resulting surfaces were then inverted into the targeted BMSs by injection molding of polystyrene (surface parameters see Table 1). Our approach was validated by employing spatiotemporal analysis, which correlates geometrical information with cellular behavior. The BSCR, not necessarily radial-symmetrical, was able to elicit in situ reversion of primed PSCs to a sustainable naive state. The PSCs on BSCR+ motifs presented apical constriction and enhanced E-cad / RAC1 signaling, with weakened cell-substrate interaction. The propagation of physical cues from BSCR interface to nuclei through a series of force dependent elements led to histone modification (H3K27me3 and H3K4me3) at promoter regions of Nanog, Zic2 and at distal enhancer region of Oct4. Topographical cues abstracted from nature blueprint offer inherent stability and robustness, which can be easily integrated into PSC substrate for large-scale implementation in bio-manufacturing processes. Bioinspired design and characterization of BMS

[0122] We analyzed the architecture of blastocysts (embryonic day, E4.25-4.5) and determined the dimensional limits of nPSCs in Epi (height: 21±9 pm, width: 59±10 pm), as well as the curvature K of the Epi / TE interface (BSCR), ranging between 15 and 62 mm-1(Fig. 1a, and Fig. 17).

[0123] To grasp both in silico and in the real-world 2.5D BMS topographies with respect to blastocyst curvature properties, we calculated and mapped the spatial information of the BMS surface based on the voxel datasets from micro computed tomography (micro-CT) (Fig. 18 a, b). Of note, we developed a scaled three-point approach (Fig. 1b,c, and Fig. 19) to measure the asymmetrical curvatures in 18 discrete directions around each surface point on the substrate. A given point was set to a radial distance of 30 pm, i.e. half the ~60 pm Epi width, to match the blastocyst scale (Fig. 1a, and Fig. 17d).

[0124] The morphological features of BMS, i.e. Convex, Flat, Concave, were then distinguished based on the mean value of the curvatures, <K>, in 18 directions. The individual curvature value, K, was used to define the BSCR+ area, in which the given point has at least one curvature within the BSCR (Fig. 1d, and Fig. 18 c-e). The representative image of a BMS shows about 8% of the BSCR+ points. The maximum BSCR count lies between 4-5. The high proportion of BSCR count at 18 directions indicated the relatively higher proportion of bowl-like structures on the substrates. The BSCR count and morphological features correlate in an intricate way, with the higher number of BSCR located on Concave motifs (Fig 1d).

[0125] Establishment of such topographical maps enabled identification and investigation of specific surface features, including the aforementioned “motifs”. Correlating these maps with microscopy images showing nPSC distribution (Fig. 20), we were able to analyze the in situ effect of geometrical cues on PSC reversion (Fig. 1e).

[0126] BSCR reverts PSCs from primed to naive state

[0127] To obtain primed cells, the PhiC31 and iPS-MEF-Ng-492B-4 mouse PSCs were chemically adapted for at least 3 passages to the primed-like state by using 1 i medium containing GSK3P inhibitor and AXIN stabilizer (Fig. 21), followed by seeding on laminin-coated BMS (Fig. 22). Compared to the Plain substrate (laminin-coated polystyrene without microstructure), PSCs on BMS exhibited the upregulated naivety genes (Fig. 7a), with NANOG, STELLA, OCT4 and SSEA1 proteins at similar levels as the naive-control (TCP, 2i / L) (Fig. 2a). For SSEA4- (a negative marker of mouse PSC) population, the percentage of SSEA1+ (a naive marker of mouse PSC) cells was higher on the BMS (94 ± 1%) than Plain substrate (89 ± 1%) (Fig. 7 b, c). Contrarily, lineage specific gene expression was decreased on BMS (Fig. 7d). ZIC2, which drives naive-to-primed transition, was downregulated (Fig. 2a).

[0128] To correlate in situ effect of detailed structural cues on PSC naivety, we sought to discriminate those nPSCs (NANOGhiand STELLAhi) using a fluorescence threshold based on the mean fluorescence intensity on a Plain control (Fig. 23) and overlaying those signals with the motif and BSCR maps (Fig. 2b, and Fig. 20). The number of nPSCs was remarkably higher in Concave motifs, than in Convex and Flat at day 1 and 3 (Fig. 2b, and Fig. 7e, f). Comparison of Concave areas with and without BSCR revealed that BSCR+ motif, rather than Concave itself, played the pivotal role to revert the naivety of PSCs (Fig. 2b). These results were supported by analyzing NANOGhiand STELLAhicells on different motifs with varying BSCR counts. At day 1 and 3, a significantly higher percentage of nPSCs was found on BSCR+ compared to BSCR- motif (Fig. 2c-d), and the naivety of nPSCs was correlated with the BSCR counts (Fig. 2e-f, and Fig. 7g). Notably, even the motifs with BSCR in only one direction (BSCR 1) was sufficient to increase the naivety (Fig. 7h-i), indicating that BSCR is the key naive pluripotency associated geometrical parameter.

[0129] To validate the in situ primed-to-naive reversion, rather than other mechanism such as cell selection and migration, we investigated the cell-substrate interaction from the initial cell attachment. After seeding, the cells exhibited random distribution on different motifs of the BMS, with similar density, NANOG expression and cell size 9 hours post cell seeding (Fig. 3a- d). The BSCR motif did not affect the necrosis of the PSCs. The apoptosis level of PSCs in BSCR+ area remained similar to BSCR- on day 0, but increased to a significantly higher level on day 3 (Fig. 8a, b). This might be attributed to the higher sensitivity to cellular stress in naive PSCs than their primed counterparts, as a result of elevated mitochondrial priming. In addition, the high cell confluence in the BSCR+ area, resulting from enhanced proliferation of nPSCs, could trigger apoptotic cell death.

[0130] To evaluate the reversion kinetics, we utilized time-lapse imaging to track live cells using a PSC line with NANOG-GFP reporter. The gradually increased GFP signals was observed in BSCR+ area as early as 21 hours after cell seeding (Fig. 3e, and Fig. 8c). By analyzing the migration of single cells, we further confirmed the in situ reversion within BSCR+ motif. From day 0 to day 1 , the migration distance of the cells on BMS was limited to 10 pm. From day1 to day 3, rare migration was observed from BSCR- to BSCR+ (Fig. 3f). These results excluded the possibility that nPSCs in BSCR+ are from BSCR- as a result of remote migration. Notably, more nPSCs was found in BSCR- area at later time point (Fig. 3e), which is in consistence with the results that similar percentage of nPSCs were found in different motifs at day 4 (Fig. 2c-d, and Fig. 7e-f). Analysis of trajectory video revealed that these nPSCs in BSCR- area are mainly from the BSCR+ area, with high confluence and subsequent expansion / migration (Fig. 3f, and Fig. 8d). The impact of motifs on PSC naivety reversion and proliferation was further examined with time-resolved imaging analysis (Figs 9 and 10). In consistence with the reported results, the naive cells (2i / L-PSCs) proliferated faster than the primed counterparts (1 i-PSCs) and the naive / primed mixed-PSCs, showing the highest increasing rate of cell density on all the tested motifs. BSCR+ motif could revert primed cells to a more naive state. For the 1 i-PSCs and mixed PSCs, which contains the primed cells, a significant increase of GFPhicell percentage was observed 21 hours post cell seeding in the BSCR+ area, compared to the BSCR- and Plain substrate. However, the percentage of Ki67+ cells remained a similar level at / before this time points. This result indicated that the increased number of naive cells in BSCR+ area at the early stage is mainly attributed to the reversion rather than the proliferation of naive cells.

[0131] Next, to verify the effect of BSCR on naivety reversion, we created the substrates with uniform microbowls (radial symmetry) and microgrooves (axial symmetry) (Fig. 11, Table 2-3). In consistence with BMS, the reversion correlate with BSCR counts, regardless the microstructure shape. The microbowls without BSCR, despite its similarity in shape to blastocyst, demonstrated a lower reversion capacity than the microgrooves with BSCR (Fig. 12a,b). The uniform microstructures with higher BSCR counts induced higher levels of naivety (NANOG- GFP, SSEA1 , TBX3, and STELLA) (Fig. 12). When PSCs were cultured on substrates with microbowls, with and without BSCR, they showed similar proliferation profile from day 1 to 3 (Fig. 12f-h), but the higher percentage of GFPhiPSCs could be found as early as day 1 on microbowl-containing BSCR (Fig. 12b). This observation is in line with that on BMS, suggesting that the increase of naive cells in BSCR+ area at the early stage is not from the proliferation, but is a result of naivety reversion.

[0132] BSCR reverts PSC naivety via E-cad / RAC1 signaling, YAP activation and histone modification

[0133] PSCs inside BSCR+ area presented morphological characteristics of naive cells, with compacted colony and decreased cell interspace along the direction of BSCR (Fig. 17f and Fig. 13a,b). The mere increase of cell density on Plain substrate did not increase NANOG level (Fig. 13c), suggesting the indispensable role of BSCR curvature on naivety reversion.

[0134] It is known that the preservation of PSC naivety can be strengthened by cytoskeleton-bound cell-cell junctions. Here, cell clusters in BSCR+ area displayed strong apical F-actin and E-cad compared to BSCR- area (Fig. 4a, b). At population level, E-cad and its stabilizer RAC1 , were upregulated on BMS (Fig. 4c, d). The inhibition of E-cad and RAC1 significantly decreased BSCR mediated naivety reversion, on both BMS (Fig. 4f-i) and substrates with uniformed microbowls (Fig. 12c). Furthermore, the nPSCs rapidly lost their naivety upon E-cad and RAC1 inhibition, suggesting the essential role of E-cad / RAC1 signaling in the preservation of naivety (Fig. 4j). We further investigated the intracellular components related to mechanical force propagation. A strong upregulation of pMLC2 and a higher cell contractility were observed in BMS-PSCs, which was abolished upon E-cad neutralization (Fig. 13d-g), suggesting the intermediary role of cell-cell adhesion in force transmission. PSCs on BMS exhibited a weakened cell-substrate interaction, with about 20% decrease of active FAK (phosphorylated / total FAK ratio) (Fig. 4e), which is in agreement with the previous report that concave curvature weakened cell-substrate interaction. The naive markers NANOG, STELLA, and primed marker ZIC2 in BMS-PSCs were not altered following FAK inhibition (Fig. 4f). These results suggested the naivety of BMS-PSCs indispensably relied on cell-cell adhesion and E-cad / RAC1 signaling, but not on FAK-mediated cell-substrate adhesion.

[0135] These findings led us to further examine the reversion of human PSCs, which might require different intracellular signaling compared to mouse PSCs. Similarly, BSCR+ motifs efficiently reverted human PSCs to a naive state (Fig. 14a), with enhanced naive markers CD7, NANOG, and reduced prime markers CD24, CD57, CD90 (Fig. 14b). Cells were more compacted on BMS than on plain substrate, showing smaller cell size (Fig. 14c) and strongly activated E-cad (Fig. 14d). The level of NANOG was significantly reduced upon inhibition of E-cad, RAC1 and YAP, but not affected by FAK inhibition (Fig. 14e). These results might indicate the similarity in key components for curvature sensing and naivety reversion between mouse and human PSCs, highlighting the feasibility of BMS for applications in human PSCs.

[0136] E-cad / RAC1 signaling can further mediate activation of YAP, which regulates stem cell fate and pluripotency as a mechanotransducer. Compared to Plain group, the total YAP level was unaltered, while inactive phosphorylated YAP was significantly reduced in BMS-PSCs (Fig. 5a). PCR array analysis showed that YAP downstream target genes were upregulated, while upstream suppressor genes were downregulated on BMS (Fig. 15a). Particularly, phosphorylated AmotLI , an inhibitor of YAP, decreased in BMS-PSCs (Fig. 15b). Given the central role of YAP in cell cycle progression, we analyzed cell cycle and found a naivety- featured rapid G1 / S transition, a higher proliferation rate and clonogenic potential in BMS-PSCs (Fig. 15c-e). Inhibition of RAC1 reduced YAP activity on BMS (Fig. 5a) and resulted in decreased cell proliferation (Fig. 15d), suggesting the involvement of E-cad / RAC1 signaling in YAP activation. The role of YAP on PSC naivety was further validated via inhibition of YAP, which significantly decreased NANOG and STELLA, while upregulated ZIC2 (Fig. 5b, and Fig. 12c).

[0137] Further, we performed ChlP-PCR to gain a better understanding of YAP in epigenetic regulation of pluripotency genes. BMS-PSCs showed significantly decreased H3K27me3 but increased H3K4me3 occupancy on “naive loci” including Nanog promoter and Oct4 distal enhancer. There were increased H3K27me3 on the promoter region of Zic2 in BMS-PSCs (Fig. 5c). YAP inhibition significantly decreased the level of H3K4me3 on Nanog promoter and Oct4 distal enhancer as well as the level of H3K27me3 on Zic2 promoter (Fig. 5c, d), suggesting that YAP activation in BMS-PSCs played a key role in histone modification of pluripotency genes. These data suggest a mechanistic relationship between BSCR, enhanced E-cad / RAC1 signaling and YAP activation, which consequently increased permissive versus repressive histone patterns in line with a gain of naivety (Fig. 5e).

[0138] BSCR stabilizes PSC naivety

[0139] To determine how long the mechanical memory and naivety can persist after the removal of BMS, nPSCs were harvested from BMS and re-seeded or injected to demonstrate the sustainable effect of BSCR regulation (Fig. 6a). The upregulated E-cad expression and YAP activity of BMS-PSCs remained for at least 15 days after reseeding (Fig. 6b-d). The enhancement of NANOG by BSCR was preserved for at least 10 days (Fig. 6e). The newly formed colonies from BMS-PSCs were more compact, with a higher proliferation rate than those from the Plain substrate (Fig. 16a-b). Larger embryoid bodies (EBs) were generated from cells pre-conditioned on BMS (Fig. 6f,g), retaining a higher E-cad level and YAP activity, as well as an accelerated cell cycle progression (Fig. 6h, and Fig. 16 c-e). To exclude the potential influence of EB size on YAP, we generated EBs with controlled dimension, and a higher YAP activity was still retained in EBs derived from BMS-PSCs (Fig. 6i). Teratoma formation was performed to determine in vivo proliferation and pluripotency of PSCs. Although cells from both groups were able to form teratomas containing three germ layers, BMS-PSCs were able to develop into teratomas with larger volume (Fig. 6j,k), suggesting their higher development potential / capacity acquired from BMS.

[0140] Discussion

[0141] In nature, although the naive PSCs only transiently exist in the blastocyst, the topographical features of a blastocyst (e.g., Epi / TE curvature) may still provide unlimited inspiration. Here, learning from nature, we explored the functionality of topographical structures derived from blastocysts on reversion of PSCs back to a naive state. We established a method to define specific surface topographical maps, and analyzed in situ the complexed microstructures on PSC reversion by correlating with time-resolved microscopy images. We concluded that the curvature constraint in single direction enabled the successful cell reversion through activation of cell-cell cohesion, intracellular signaling and epigenetic modulation, without necessarily recapitulating the relative radial-symmetry of blastocyst.

[0142] We demonstrated that the reverted naivety of PSCs was sustained for at least ten days after removal of BMS. The BMS-PSCs processed the higher development potential in generating embryoid bodies and teratomas, as compared to cells on non-structured substrates.

[0143] The blastocyst scaled curvature range (BSCR) reported here is inspired and simplified from nature, but going beyond nature, which provides the design criteria for preparing functional substrate with a purpose to achieve high sustainability in stem cell applications. Importantly, our results indicated the possibility of the BSCR for reverting human PSCs to a more naive state. Although the underling mechanism of human PSCs should be further explored, the results highlighted the importance of substrate design in reversion of human PSC naivety and the profound applications, such as organoid generation, drug screening, disease models and personalized medicine.

[0144] Methods

[0145] Determination of mouse blastocyst geometry

[0146] The confocal cross views of preimplantation blastocysts (E4.25-4.5) from literatures were imported to Imaged software with Bio-Formats Importer plugin (National Institutes of Health). The width of naive pluripotent stem cell (PSC) layer was measured by detecting the line segments between two endpoints of epiblast (Epi) in contact with polar trophectoderm (TE) (Fig. 7a-b). The distance from the midpoint of the width line to the Epi-TE interface was defined as a height of the outer layer of Epi. The radii of the arcs, curvatures, arc lengths and cell densities of the outer layer of Epi were calculated based on the following formulas: arc radius = width2 / (8 x height) + height / 2; curvature = 1 / arc radius; arc length = 2 x radius x arcsin (width / (2x radius)); cell density = cell number / arc length. The polar TE layer provides a relatively well- defined curvature of 15 to 62 mm-1(31 ± 8 mm-1, mean ± SD), which we refer to as the blastocyst-scaled curvature range (BSCR).

[0147] Defining motifs on cell culture substrates

[0148] The scaled three-point approach (see supplementary method for details) was used to measure the curvature of a given point on the BMS in 18 directions. The curvature value of a single direction, K, and the mean curvature values of 18 directions, K , were used to define the motif regions on BMS. Here, we define a point as BSCR+ if at least one curvature, K, for that point is within the BSCR (15 mm-1< K < 62 mm-1). Next, we distinguished regions, which have a positive mean curvature K value, i.e. Concave, and negative K value, i.e. Convex. Between both Concave and Convex regions we defined a Flat region, with some tolerance for positive and negative average curvature values. The regions were defined in detail as follows: Convex ((K) < -2.5 mm-1), Flat ( -2.5 mm’1< (K) < 2.5 mm-1), Concave ((K) > 2.5 mm-1).

[0149] Validation of motif function on PSC naivety reversion

[0150] The bright field microscopic images of the BMS were first recognized to be precisely overlaid onto the micro-CT scanning images. The defined BSCR+ area, Concave, Flat, and Convex motifs based on the micro-CT scanning and curvature calculation were then mapped and overlaid with the microscopic images to evaluate the effect of geometrical motifs on cells. In brief, the maximum intensity projection CLSM images of cells and the motif maps in the same region were imported into Photoshop software (Adobe Systems), and the motif maps were used as a mask to study the cellular behavior and parameters on defined areas, including cell size, density, distribution, migration, naive marker level. Live cell tracking

[0151] NANOG reporter-GFP, which integrated in iPS-MEF-Ng-492B-4 cells (CiRA, Kyoto University, Japan) was detected by Argon channel (488 nm excitation) to track the living mouse PSCs. To monitor naivety reversion and cell migration, real-time fluorescence images of PSCs and bright field images of BMS surface in Z-stack were acquired using CLSM (Carl Zeiss). PSC expansion on BMS in a single colony were recorded at 20 min intervals using time-lapse fluorescent microscope (1X81 , Olympus).

[0152] For image analysis, maximum intensity projection was first performed for all the z-stack CLSM images. The GFP intensity of cells was analyzed using Imaged software (National Institutes of Health). The maximum GFP intensity of cells on Plain substrates was set as a threshold. On BMS, the cells with higher intensity than such a threshold were defined as GFPhinaive PSCs. MountainsMap® (Digital Surf, Besancon) and Python 3.6 software were then used to analyze the effect of motifs on GFPhicell occupancy, naivety level in a time-resolved manner.

[0153] E-cad, RAC1, FAK, YAP activity and inhibition

[0154] To examine the distribution and expression of E-cad and F-actin with respect to motifs, cells were fixed, permeabilized and blocked using Image-iT Fixation / Permeabilization Kit (Life Technologies). anti-E-cad-PE antibody (rabbit monoclonal; 1 :10; New England Biolabs GmbH) was used for labelling E-cad. F-actin was detected using ActinRed 555 ReadyProbes Reagent (Life Technologies). The CLSM images of the samples were then overlaid with motif maps following the method shown in Fig. 20.

[0155] To quantify E-cad level, 1x106cells were freshly harvested from the substrates at indicated time points, then immediately processed for anti-E-cad-PE antibody (rabbit monoclonal; 1 :50; New England Biolabs GmbH) staining in dark at room temperature for 30 minutes. The labeled cells were measured by flow cytometry (MACSQuant, Miltenyi Biotec.) and analyzed using “Flowjo” software (Tree Star Inc.).

[0156] The concentration of phosphorylated FAK and total FAK in the cell extract were measured using the pFAK (pY397) / tFAK ELISA kits (Thermo Fisher Scientific). The same amount of total protein determined using a BCA protein assay kit (Thermo Fisher Scientific Inc.) was applied for ELISA. For quantification of YAP and AM0TL1 phosphorylation levels in PSCs, day-3 cells growing on different substrates were harvested, fixed with 4% paraformaldehyde and permeabilized with pre-chilled 90% methanol for 30 minutes on ice, then stained with anti -YAP- Alexa fluor 647 and anti-phospho-YAP (S127) (rabbit monoclonal; 1 :50; New England Biolabs GmbH), or anti- phospho-AMOTLI (S262) antibodies (rabbit polyclonal; 1 :100; Covalab Biotechnology) for 45 minutes. The cells were then stained with secondary anti-rabbit IgG (H+L)-Alexa fluor 647 and anti-rabbit IgG (H+L) Alexa fluor 488 antibodies (goat polyclonal; 1 :500; New England Biolabs GmbH) for 30 minutes, followed by flow cytometry analysis.

[0157] Inhibition experiments were performed by treating the cells using the RAC1 activity inhibitor NSC 23766 (50 pM; Bio-Techne GmbH), the selective FAK inhibitor PF-573228 (10 pM; Sigma- Aldrich) and the YAP activation inhibitor Verteporfin (20 nM; Sigma-Aldrich). For E-cad neutralization, cells were treated with E-cad antibody (25 pg / ml; Merck KGaA) for 24 hours before harvesting.

[0158] Chromatin immunoprecipitation (ChlP)-PCR

[0159] The ChIP was performed using the SimpleChIP Plus Enzymatic Chromatin IP Kit (New England Biolabs) following the given instruction. Chromatin immunoprecipitations were performed using anti-H3, anti-H3K27me3 and anti-H3K4me3 antibodies (rabbit monoclonal; 1 :50; New England Biolabs) with magnetic beads. After washes, samples were eluted and treated with RNase and proteinase K overnight. The released DNA was used for the real-time PCR quantification. Enrichment was calculated relative to input DNA data and expressed as percent Input = 10 %X normalized to total H3 enrichment level. Normal rabbit

[0160] IgG (1 :50; New England Biolabs) was used as an isotype control.

[0161] Stability of naivety of BMS-PSCs

[0162] PSCs were cultured on Plain and BMS for 5 days, then harvested and re-seeded to evaluate the stability of naivety. On laminin coated TCP, the cells were seeded at a density of 2 x io4 / cm2and cultured in monolayer for 4 passages, followed by anti-E-cad-PE (rabbit monoclonal; 1 :10; New England Biolabs GmbH), anti-YAP- Alexa fluor 488 (rabbit monoclonal; D8H1X; 1 :100; New England Biolabs GmbH) and anti-NANOG-APC (recombinant human monoclonal; 1 :11 ; Miltenyi Biotec) staining. The samples were then examined with confocal laser scanning microscopy (LSM780, Carl Zeiss) and flow cytometry.

[0163] Embryoid bodies (EBs) were obtained by re-seeding the cells (2 x io4 / cm2) on un-coated TCP, and cultured in complete KnockOut Serum Replacement EB medium (Thermo Fisher Scientific Inc.) To form EBs with uniformed dimension, a 96-well V bottom plates (Sigma-Aldrich) was used. The generated EBs were fixed, permeabilized and incubated with anti-YAP (rabbit monoclonal; D8H1X; 1 :100; New England Biolabs GmbH) overnight at 4 °C, the secondary anti-rabbit IgG (H+L)-Alexa fluor 647 or anti-rabbit IgG (H+L) Alexa fluor 488 antibodies (goat polyclonal; 1 :500; New England Biolabs GmbH) was used for labeling. After 1 hour culture at room temperature, the fluorescence was detected and imaged by confocal laser scanning microscopy (LSM780, Carl Zeiss). The YAP phosphorylation level was examined using Western blot (see Supplementary Method below for details).

[0164] The teratoma formation experiment was performed by EPO GmbH (Experimental Pharmacology and Oncology, Berlin-Buch) and carried out in accordance with the German Animal Protection Law and approved by the local responsible authorities. EPO complies to the Ell guideline “European convention for the protection of vertebrate animals used for experimental and other scientific purposes (ETS 123)”. Animals were handled according to the “Regulation on the protection of experimental scientific purposes or other Purposes used animals”. Compliance with the above rules and regulations is monitored by the Landesamt fuer Gesundheit und Soziales (LAGeSo) which is the responsible regulatory authority monitoring the animal husbandry based on the German Animal Welfare Act. Approval (code: H 0023 / 09) was given after careful inspection of the site including bedding, feeding & water, ventilation, temperature, and humidity, cleaning and hygiene concepts. The day-5 PSCs derived from Plain and BMS substrates were subcutaneously injected into 8-week old male NSG immunodeficient mice (1 x 106PSCs for each). The teratoma diameter measurements were performed twice weekly until the end day of the study when teratoma reached 1.5 cm3. Teratoma volume was calculated using the formula: Volume = Width2x Length / 2. The dissected teratomas were cryoconserved or formalin-fixed for haematoxylin and eosin staining.

[0165] Statistical analysis

[0166] Statistical analysis for biological evaluation was performed using Prism Graphpad 8.0 (GraphPad Software). Comparisons between two experimental groups or the same group with and without inhibitors, were analyzed using two-tailed unpaired sample Student’s t-tests. Prior to perform the Student’s t-test, the homogeneity of variance for the two groups was verified by F-test. The differences among three or more independent groups were analyzed using one- or two-way ANOVA followed by Bonferroni’s multiple comparisons test. Particularly, the two-way ANOVA was applied to analyze the individual effects of motifs and BSCR counts on regulating cell distribution, proliferation and naive markers as well as their overall effects over time. The sample sizes for each experimental group and the statistical test used to determine significances among groups are reported in the figure legends. Briefly, for the following in vitro and in vivo studies: quantification of naive pluripotency associated markers, E-cad, FAK, YAP, histone H3 modification at naive gene loci, cell death, migration, cell cycle sub-phases, cell expansion and Teratoma formation, statistical analyses were applied to three biologically independent samples (separate batches / plates of cells, cell lysates and immunodeficient mice) across Plain and BMS. For image-based analysis with Imaged software (National Institutes of Health), the numbers of images, cell nuclei and colonies were indicated in the figure legends accordingly. In all cases, p values of less than 0.05 were considered statistically significant and indicated using different thresholds (p < 0.05, 0.01 , 0.001 , 0.0001). Unless indicated otherwise, the quantification data are presented as mean ± standard deviation.

[0167] Supplementary Methods

[0168] Fabrication of BMS

[0169] Four metal cylinders (steel type 1.2210, 60 HRC) were prepared using the electrical discharge machining (EDM). For the spark erosion process an EDM machine of model Gantry Eagle 500 (OPS-INGERSOLL Funkenerosion GmbH, Burbach, Germany) was applied utilizing an electrode type graphite EDM 200 (Poco Graphite SARL, Entegris, Inc., Limonest, France) and lonoPlus IME MH (Oelheld GmbH innovative fluid technology, Stuttgart, Germany) as dielectric medium. The machine has an adaptive current pulse generator Eagle Powertec Fine Finishing generator PT60 with maximum machining power 60 A. The estimated average energy per spark is 5.4 - 47.7 pJ. Machine parameters were used as following: work piece material: Steel; electrode material: Grs Graphit standard; electrode geometry: Standard (free form); engagement area: 78.0 mm2; cross section area: 78.0 mm2; erosion depth: 25 mm; and priority: low-wear. Each metal cylinder was machined for 30 minutes to achieve a surface finish grade M45 according to the guideline VDI 3400 surface finish grade definition. This corresponds to a surface roughness of Ra ~ 17.8 pm and a maximum distance between the highest peak and lowest valley in the whole sampling of Rt - 110.0 pm. Electrode-Typ 1 was used with an undersize distance of 0.3 mm. With this roughness, blastocyst relevant dimensions were reached. Finally, the eroded metal cylinders were intensively purified utilizing PowerCleaner 200 (Bio-Circle Surface Technology GmbH, Gutersloh, Germany).

[0170] Polystyrene (PS, type 158K, BASF, Germany) with a number average molecular weight of Mn= 109 000 g mol-1was used without any further purification. Processing of the BMS polymer inserts fitting into standard 24-well tissue culture plates was performed by injection molding using an injection molding automat (Alrounder 270LI, Arburg Corp., Munsingen, Switzerland) equipped with a custom-made mold (Dreuco Formenbau GmbH, Berlin, Germany). In a two- stage process a volume 6.5 cm3PS was injected with injection rates of 14 and 20 cm3-s'1and pressures of 1200 and 1800 bar. The applied temperatures were 35 °C (heating zone I), 210 °C (heating zone II) and 230 °C (heating zone III and IV), while the mold was kept at 30 °C. The molds consisted of the above mentioned four individually surface-finished metal cylinders, allowing the parallel fabrication of four inserts (BMS1-BMS4) with an upper inner diameter of 12.4 mm, a lower inner diameter of 10.5 mm, a height of 16.8 mm, and a wall thickness of 1 mm.

[0171] Prior to use, all prepared PS inserts were sterilized by ethylene oxide gas sterilization (gas phase: 10% ethylene oxide, 54 °C, 65% relative humidity, 1.7 bar, 3 hours of gas exposure time and 21 hours of aeration phase).

[0172] Fabrication of microbowl and microgroove substrates

[0173] PS based microbowl (pB) and microgroove (pG) substrates were prepared to study the effect of regular micro-structure on PSCs.

[0174] Acid washed glass beads (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) with diameters of -75, 150-210, and -250 pm were used to prepare the pB-substrate. These glass beads were first functionalized with silanization solution (1% dichlorodimethylsilane in ethanol, Sigma) and then rinsed with ethanol before air-dried in the hood. Polyvinyl alcohol (P A) was dissolved as 5% (w / v) solution in de-ionized water, and 5 mL of the solution was placed into a 35 mm petri dish. Silanized glass beads were then gradually dropped onto the meniscus, which floated on the liquid-air interface due to hydrophobicity, and spontaneously assembled into a single, closely packed layer under ultrasound for 30 minutes. The pG-substrates were produced by photolithography methods. 2-inch silicon wafers were spin-coated with 2 mL of ma-P 1275G positive photoresist (Micro resist technology GmbH, Berlin, Germany) at 500 rpm for 60 s, followed by 1 hour evaporation and a three-stage baking step at 70 °C, 90 °C, 115 °C for 5 minutes each. Photomasks designed by Autodesk software were printed at 128k dpi (JD Photo Data company, Hitchin, UK), placed on the coated wafer, and exposed for 80 s to UV-light irradiation (mercury arc lamp 365 nm, intensity 114 mW / cm2; Solar simulator system, Abet technology) using a UV filter (cut-on 365 ± 7 nm, Laser Components GmbH, Olching, Germany). Finally, the exposed wafers were post-baked at 100 °C for 30s and developed in mr-D 526 / S developer (Micro resist technology GmbH, Berlin, Germany) with gentle shaking. Finally, the developed silicon wafer was heated at 105 °C for 5 minutes to allow the reflow process, which converts the rectangular cross-sectional shape of photoresist to a curved shape.

[0175] The poly(dimethylsiloxane) (PDMS) soft mold replicate of the assembled glass beads or structured silicon wafer was synthesized from a precursor mixture of 90 wt% prepolymer Sylgard 184 and 10 wt% curing agent (Dow Corning Corp., Midland, Ml) by curing at 80 °C for 24 h. The achieved PDMS mold was applied as negative template for producing a second PDMS soft mold comprising microprotrusions following the aforementioned procedure. For preparing smooth surface, the PDMS soft mold was replicated from smooth silicon wafer.

[0176] Finally, the PS substrates with pB and pG microstructures were obtained by soft-lithography according to the reference2, using the prepared PDMS molds. Prior to use, the microstructured PS substrates were sterilized by ethylene oxide gas sterilization (gas phase: 10% ethylene oxide, 54 °C, 65% relative humidity, 1.7 bar, 3 hours of gas exposure time and 21 hours of aeration phase).

[0177] Micro computed tomography (micro-CT) analysis of BMS

[0178] An X-ray micro-CT of ProconXray GmbH (Germany) was used for characterization of surface texture of BMS inserts. Recording of grey shadow images was realized with an X-ray output of 40 kV and 0.2 mA at small spot. The distance between X-ray source and sample resulted in a voxel dimension of 9.1 pm edge length. The integration time was 750 ms at averaging of 6 images. Overall 960 images, recorded with an angle distance of 0.375 degrees, were used for reconstruction of 3D images. The BMS inserts were oriented perpendicular to the measurement beam. The post treatment of 3D-images was carried out with MAVI software (Fraunhofer Society, Germany). The reconstructed 3D-images (example shown in Fig. 18a) were filtered with a binomial filter stage 19 and binarized by the “Otsu” algorithm.

[0179] For 3D surface topography analysis of the PS inserts, the reconstructed surfaces from micro- CT (stored as . stl-files) were analyzed using MountainsMap® software (Digital Surf, Besancon, France) corresponding to EN ISO 25178. Initially a circular shape was cut out from the bottom of the insert, with the side walls removed, and thereafter an operator for leveling by a least square method was applied. The height-maps of the four BMSs with a diameter of 10.8 mm are shown in Fig. 18b. The images show round-shaped elevations and hollows, confirming the absence of sharp edges and vertical or horizontal planar areas. The x,y,z-data on a spacing grid of 15 pm in x- and y-direction were used for further curvature evaluation.

[0180] Scaled three-point approach for curvature measurement

[0181] The method we have used in this invention we label scaled three-point curvature and is based on Heron’s formula. On a surface S for any given point p, curvature is analyzed around the point itself 360 degrees (Fig. 1b and Fig. 19). This is done by giving a direction in the form of a vector, v, then two other points are selected by going in the positive direction of the vector and the negative direction of the vector. Once the three points are selected, a system of equations can be written to solve for a circle that touches all three points. The inverse of the radius is the curvature for that point p in the direction v. This is then repeated for n directions for each point. We chose to compute curvature, K, at each 10-degree increment, yielding 36 curvatures. However, due to symmetry only 18 are unique. The magnitude of vector, v, is the scale, with which the curvature was analyzed. The magnitude for v was set to 30 pm, as this reflected half the width of a Epi which is ~60 pm (Fig. 19).

[0182] Laminin (LMN) coating and characterization

[0183] To facilitate mouse PSC attachment, Plain and BMS substrates were pretreated with 200 pl of 9 pg / ml Cultrex Mouse Laminin (Bio-Techne GmbH). Anti-laminin antibody (rabbit monoclonal; 1 :100; Abeam) was used for LMN immunostaining. The distribution of LMN on different substrates (20 images) and in different motif areas (10 images) was quantified via the confocal image-based analysis of LMN intensity using imaged software (National Institutes of Health). The LMN formed a homogeneous layer without strong difference in coating density on BMS and Plain control, as well as between different motifs. The amounts of LMN in the coating solution at day 1 and released in PBS from day 2-5 on different substrates were quantified using LMN ELISA Kit (abeam). Three independent experiments were included. The LMN solution was collected daily and the LMN adsorption amount was calculated by subtracting the soluble LMN from total LMN amount at the previous day. The amount of LMN remained to be constant for 5 days, indicating the stability of the LMN layer. Further, the advancing and receding contact angles of LMN coated and uncoated substrates were measured with drop shape analyzer (DSA 100, Kruss GmbH) using the captive bubble method. All samples were pre-conditioned for 24 hour in deionized water at ambient temperature for equilibration. 10 measurements for advancing and receding angles on five different locations were performed for each sample. LMN coating increased the wettability of the substrates.

[0184] Cell culture

[0185] The non-viral integrating mouse PSC cell line (PhiC31 , BioCat GmbH, reprogrammed from C57BL / 6 embryonic fibroblasts (MEFs) with a plasmid encoding OCT4, SOX2, KLF4, and c- Myc) was used for assessing the reversion to naivety. The mouse cell line with NANOG- reporter GFP (iPS-MEF-Ng-492B-4 cells, CiRA, Kyoto University, Japan) was used for live cell tracking. The initial culture of both mouse PSC cell lines was performed using mitomycin C treated feeder MEFs (BioCat GmbH) and 0.2% gelatin coated culture plate, in feederdependent PSC culture medium (KnockOut DM EM basal medium containing 15% KnockOut Serum Replacement, 1% MEM Non-Essential Amino Acids, 1 % GlutaMAX-l, 0.1% 2- mercaptoethanol and 10 ng / ml mouse LIF, Life Technologies). Undifferentiated PSCs were isolated using magnetic-activated cell sorting (MACs) technology with Feeder Removal MicroBeads and Pluripotent Stem Cell Isolation Kit (Miltenyi Biotec), PSCs were then maintained in ESGRO-2i / L Medium (containing: GSK3P and MEK 1 / 2 inhibitors and 0.5 x 106U / ml mouse LIF, Merck Chemicals GmbH), and cultured on 9 pg / ml Cultrex Mouse Laminin (LMN) (Bio-Techne GmbH) coated 6-well tissue culture plates. The PSCs were chemically adapted for at least 3 passages to the primed-like state by using 1 i medium containing GSK3P inhibitor (3 pM CHIR99021 , Stemgent, Inc,) and AXIN stabilizer (2 pM XAV-939, Stemgent, Inc.). The 2i / L-PSCs and 1 i-PSCs maintained in tissue culture plates were used as positive and negative controls for naive cells, respectively (Fig. 20a). The culture media were changed daily and all PSCs under aforementioned culture conditions were used for experiments before reaching passage 50. All PSCs applied for experiments were detected negative for mycoplasma using MycoFluor Mycoplasma Detection Kit (ThermoFisher Scientific). The morphology of PSCs under different culture conditions was monitored, compared to the 2i / L- PSCs, the 1 i-PSCs presented primed-like characteristics, including the elongated cell shape and flattened colonies (Fig. 20b), as well as decreased levels of NANOG reporter-GFP (the mean fluorescent intensity (MFI) of GFP on Plain substrates was set as threshold) and NANOG proteins (Fig. 20c-d).

[0186] Human PSC cell line (BIHi001-A) was a gift from the Stem Cell Core Facility, Berlin Institute of Health, Germany. This exogene integration-free cell line was generated using Sendai virus vectors. Detailed information is available in the hPSCreg database (http: / / hpscreg.eu / cell- line / BI Hi001 -A). Human PSCs were maintained on Geltrex (ThermoFisher Scientific) coated TCP under feeder-free conditions using Essential 8 medium (ThermoFisher Scientific). The human PSCs were adapted for 10 passages to a naive-like stage using RseT medium (STEMCELL Technologies) in the presence of irradiated CF1 mouse embryonic fibroblasts (ThermoFisher Scientific) under hypoxic condition (5% O2). The RseT-human PSCs were set as a positive control for human PSC analysis.

[0187] Confocal laser scanning microscopy (CLSM)

[0188] To measure the cell diameters, the cytosol of live PSCs was stained with 1 pM CellTrace™ Far Red (Thermo Fisher Scientific Inc.) for 15 min at 37 °C. The dead or dying cells on BMS were detected with 1 pg / ml propidium iodide solution (Miltenyi Biotec.).

[0189] To investigate the effect of BSCR on PSC proliferation, iPSCs (iPS-MEF-Ng-492B-4 cells with integrated NANOG reporter-GFP, CiRA, Kyoto University, Japan) with different initial state (Primed, naive, and mixed population (naTve:primed = 1 :4 in number)) were seeded on BMS substrate as well as Plain surface at a seeding density of 2x104cells / cm2. Subsequently, primed and mixed PSCs were cultured in 1i medium, while naive PSCs were cultured in 2i / L medium. Ki67 and DAPI staining were carried out at specified time points for comprehensive analysis.

[0190] For intracellular and intranuclear imaging, cells were fixed, permeabilized and blocked using Image-iT Fixation / Permeabilization Kit (Life Technologies). Click-iT™ Plus TUNEL Assay Kit (Thermo Fisher Scientific Inc.) was used for in situ apoptosis detection with Alexa fluor 647 dyes. Cells were stained with primary antibodies overnight at 4 °C and were then incubated with corresponding Alexa fluor 488 conjugated anti-mouse or rabbit IgG (H+L) secondary antibodies (1 :800; Thermo Fisher Scientific Inc.), anti-mouse IgG (H+L)-Alexa fluor 647 secondary antibody (1 :500; Thermo Fisher Scientific Inc.) and anti-rabbit IgG (H+L)-Alexa fluor 633 secondary antibody (1 :800; Thermo Fisher Scientific Inc.) for 1h at room temperature in the dark. The following primary antibodies were used: anti-NANOG (rabbit polyclonal; 1 :10; Thermo Fisher Scientific Inc.), anti-STELLA (mouse monoclonal; 1 :50; Merck KGaA) and anti- TBX3 (rabbit polyclonal; 1 :200; Thermo Fisher Scientific Inc.), anti-Ki67 antibody (rabbit monoclonal 1 :400; New England Biolabs GmbH) and rabbit IgG isotype control antibody (Thermo Fisher Scientific Inc.). The viable and fixed cell nuclei were stained with Hoechst 33342-based NucBlue Live ReadyProbes (Life Technologies) and DAPI (Thermo Fisher Scientific Inc.), respectively. CLSM imaging was carried out using LSM 780 (Carl Zeiss). The stitched tile scanning (7 mm x 7 mm) and the local scanning (1.4 mm x 1.4 mm and 1.4 mm x 0.7 mm) of z-stack images of NANOG, STELLA and the bright field view of PSCs on BMS at indicated time points were recorded. The cross views were reconstructed using Zen 10 software (Carl Zeiss) or “dynamic resliced” function of Imaged (National Institutes of Health). Maximum intensity projection was performed for all the z-stack image series of cells on Plain and BMS. The maximum fluorescence intensity of NANOG and STELLA in cells grown on Plain substrates were set as thresholds. The CLSM images of NANOGhiand STELLAhicells used for the following motif and BSCR analysis were obtained by applying the thresholds, respectively (Fig. 23). To study the cell proliferation activity of PSCs, a set of fluorescence images, including GFP, Ki67, nuclei, and bright-field images in Z-stack, was acquired. A maximum intensity projection was subsequently carried out for analysis. The threshold for the GFP filter was set using the maximum GFP intensity of 1 i-PSCs on the Plain substrate, discriminating between GFPhiand GFP|OWcells. Ki67+ cells were defined using the maximum intensity of the Ki67- isotype control. The BSCR mask, derived from micro-CT analysis, was employed to differentiate between BSCR+ and BSCR- areas. The GFPhi-cell covered area within the BSCR+ region served to create the mask for GFPhi / BSCR+. For precise cell identification, Otsu’s Local Thresholding and Watershed Separation were applied through image segmentation. Imaged Macro programming (National Institutes of Health) was utilized for the automation of image analysis, as illustrated in Fig. 24.

[0191] Flow cytometry To analyze the mouse PSC naivety, 1x106cells were freshly harvested at indicated time points, then immediately processed for flow cytometry analysis of surface markers. The fixed and permeabilized cells were used for analyzing of intracellular proteins. Cells were incubated directly with anti-stage-specific embryonic antigen (SSEA) 1-PE (mouse monoclonal; 1 :50; Thermo Fisher Scientific Inc.) and anti-SSEA-4-Alexa fluor 647 (mouse monoclonal; 1 :10; BD Biosciences), anti-NANOG-APC (REAfinity recombinant human IgG; 1 :11 ; Miltenyi Biotec.), anti-OCT4-PE (REAfinity recombinant human IgG; 1 :11 Miltenyi Biotec.), anti-STELLA (mouse monoclonal; 1 :50; Merck KGaA) and anti-ZIC2 (rabbit monoclonal; 1 :600; abeam) antibodies in dark at room temperature for 30 minutes. The primary antibody solution of anti-STELLA and anti-ZIC2 was removed. Cells were then incubated with anti-mouse and anti-rabbit IgG (H+L)- Alexa fluor 647 antibodies (goat polyclonal; 1 :500; New England Biolabs GmbH) for another 30 minutes, respectively. Labeled cells were measured by flow cytometry (MACSQuant, Miltenyi Biotec.) and analyzed using “Flowjo” software (Tree Star Inc.). For human PSC measurement, the following antibodies were used: anti-CD7-APC (REAfinity recombinant human IgG; 1 :50; Miltenyi Biotec.), anti-CD24-APC (REAfinity recombinant human IgG; 1 :50; Miltenyi Biotec.), anti-CD57-FITC (mouse IgM monoclonal; 1 :50; Miltenyi Biotec.), anti-CD90-FITC (REAfinity recombinant human IgG; 1 :50; Miltenyi Biotec.), anti-E-Cadherin-PE (rabbit monoclonal; 1 :50; New England Biolabs GmbH) for living cells, and anti-NANOG-APC (REAfinity recombinant human IgG; 1 :11 ; Miltenyi Biotec.) for fixed / permeabilized cells.

[0192] To analyze the alterations in cell cycle kinetics, the PSCs were seeded with the density of 1.5 x 104cells / cm2. Day 3 PSCs were harvested using accutase (Merck KGaA) and fixed overnight with cold 70% ethanol at 4 °C and subsequently stained with FxCycle PI / RNase Staining Solution (Life Technologies) at room temperature for 30 minutes. DNA content was estimated by flow cytometry, and the fractions of cells in different phases were analyzed using “ModFit LT” software (Verity Software House). The same procedure was applied for EBs derived from PSCs pre-conditioned on different substrates. Accumax (Merck KGaA), Embryoid Body Dissociation Kit and gentleMACS Dissociator (Miltenyi Biotec.) were used for EB detachment and dissociation. Single cell samples were applied for all the flow cytometric measurements (MACSQuant, Miltenyi Biotec.).

[0193] Real-time PCR Array

[0194] RNA was extracted and purified from the day 3 PSCs using the RNeasy Mini Kit (QIAGEN GmbH), and was subsequently transcribed into cDNA with the RT2 First Strand Kit according to the manufacturer's protocol (QIAGEN GmbH). Real-time PCR array analysis was performed using mouse embryonic stem cell and mouse Hippo signaling pathway RT2 Profiler PCR Array microplates (QIAGEN GmbH) on a StepOne Plus Real-time PCR System (Applied Biosystems). Data were uploaded and analyzed with free online RT2 Profiler PCR Array Data Analysis Web software (QIAGEN GmbH). The cut-off was set as CT (threshold cycle) values higher than 35, which considered as absence of gene expression. The CT value of mouse heat shock protein 90 kDa alpha, class B member 1 (Hsp90ab1) housekeeping gene in each sample was used to normalize the ACT values of target genes (ACT = CT, target - CT, housekeeping). The gene expression level was expressed as 2-ACT, and the fold change between two samples (Sample 2 / Sample 1) was expressed as 2-AACT(AACT = ACT, target, Sample 2 - ACT, target, Sample 1).

[0195] Western blot

[0196] 2 x io4 / cm2pre-conditioned PSCs from Plain and BMS were reseeded to the 6-well suspension culture plate to form EBs. At day 3, EBs were lysed at room temperature for 10 minutes with M-PER Mammalian Protein Extraction buffer containing a mixture of 1 x Halt Protease & Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific Inc.). The protein concentration in the supernatant was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific Inc.). 20 pg of total protein from each sample was loaded to the AnykD precast SDS-PAGE gel (BioRad Laboratories GmbH) and electrophoresis was performed at 150 V. When the protein dye reached the bottom of the gel, the protein was transferred onto the nitrocellulose membrane (Merck KGaA) at a constant current of 200 mA for 1 h. Then, the membrane was blocked with Odyssey Blocking Buffer (LI-COR Biosciences) and stained with anti-E-cad (mouse monoclonal; 1 :500; Thermo Fisher Scientific Inc.), anti-phospho-YAP (S127), anti-YAP and anti-GAPDH antibodies (rabbit monoclonal; 1 :1 ,000; New England Biolabs). Finally, the primary antibodies bound to the membrane were detected with IRDye 800CW secondary antibody and visualized using an Odyssey Infrared Imaging System (LI-COR Biosciences). The experiment was performed using three independent cell preparations and the protein level was quantified by analyzing the density of bands with Imaged software (National Institutes of Health).

[0197] Alkaline phosphatase (AP) staining Stemgent Alkaline Phosphatase Staining Kit II (Miltenyi Biotec) was applied for AP staining of PSCs. Cells were fixed with Fix Solution at room temperature for 5 minutes. The fixed cells were washed with PBST and incubated with freshly prepared AP substrate solution in the dark at room temperature for 10 minutes. The reaction was stopped by washing the wells twice with PBS. Cells were then covered with PBS or mounting medium to prevent drying and stored at 4°C for microscopy.

[0198] Cell proliferation assay

[0199] The PSCs were seeded with the density of 1.5 x 104cells / cm2on Plain and BMS substrates. The relative number of cells at day 1 , 3, 5 was determined using the Cell Counting Kit-8 (CCK- 8, Dojindo Molecular Technologies). In brief, old medium was replaced with 300 pl of fresh medium, followed by adding 30 pl CCK-8 solution. After 2 h of incubation at 37 °C, 100 pl medium / CCK-8 mixture was transferred into a transparent 96-well plate, and the absorbance was measured at a wavelength of 450 nm (reference: 650 nm) using a microplate reader (Infinite 200 PRO, Tecan Group Ltd.). The cell number was calculated via a standard curve, which was produced by measuring a series of samples with known cell number.

[0200] RAC1 activation G-LISA

[0201] Cells were washed and lysed in ice-cold cell lysis buffer provided in G-LISA RAC1 activation assay kit (Biomol GmbH) and immediately clarified by centrifugation at 10,000 x g, 4°C for 1 minutes. The protein concentration was determined using Precision Red Advanced Protein Assay Reagent. The powder immobilized in RAC1-GTP binding well strip was dissolved on ice for 20 minutes and 50 pl of concentration-equalized lysates, RAC1 positive and buffer blank control were then applied and incubated on a cooled orbital microplate shaker at 400 rpm, 4°C for 30 minutes. Wells were washed twice and incubated with 200 pl of antigen presenting buffer for 2 minutes. 50 pl of 1 :50 diluted anti-RAC1 antibody was loaded and incubated at 400 rpm for 45 minutes. 50 pl of secondary antibody was added and incubated at 400 rpm for 45 minutes. 50 pl of the freshly prepared HRP detection reagent (reagent A and B in equal volumes) was loaded and incubated for 20 minutes. After 50 pl of HRP stop buffer was applied to each well. The absorbance was measured at 490 nm using the Tecan Infinite 200 PRO microplate reader (Tecan Group Ltd.). pMLC2 imaging and quantification

[0202] Day 3 PSCs were directly fixed on culture substrates or detached and harvested followed by a fixation with 4% paraformaldehyde. 0.5% Triton X-100 was used for permeabilization. Cells were then stained with the primary anti-phospho-myosin light chain 2 (S19) antibody (mouse monoclonal; 1 :100; New England Biolabs GmbH) followed by the staining of the anti-mouse IgG (H+L)-Alexa fluor 647 secondary antibody (goat; 1 :500; New England Biolabs GmbH). The fluorescent intensity of pMLC2 in Concave+ and Concave- motifs was measured by LSM780 microscope (Carl Zeiss). The overall level of pMLC2 in cell grown on substrates was detected by MACSQuant flow cytometer (Miltenyi Biotec.). Data analysis was performed using “Flowjo” software (Tree Star Inc.).

[0203] Collagen matrix-based contractility assay

[0204] The working solution of collagen gel was freshly prepared and kept on ice according to the user manual of Cell Contraction Assay Kit (BioCat GmbH). Single cell suspension (2 x 106cells / ml) was collected and mixed with collagen gel (1 :4 vol / vol). The mixture was then loaded and solidified in the culture plate at 37°C for 1 hour. At day 3, the stressed gel was gently released from the side of culture well with a sterile spatula. 8 hours after releasing, the covered area change of the gel (% of initial covered area) was captured by microscope and measured by Imaged software (National Institutes of Health).

[0205] Tables

[0206] Table 1. Comparison of surface parameters between metal molds and BMS according to DIN EN ISO 25178

[0207] Property Parameter Unit Metal molds BMS

[0208] Arithmetical mean height of the surface Sa pm 19.7±0.7 20.7±2.1

[0209] Root mean square height of the surface Sq pm 25.2±0.7 26.1 ±2.0 Maximum height of peaks Sp pm 103±8 72.7±7.2

[0210] Maximum height of valleys Sv pm 68.7±8.4 105±1.0

[0211] Maximum height of the surface Sz pm 172±14 178±7

[0212] Skewness of height distribution Ssk 0.49±0.23 -0.57±0.13

[0213] Kurtosis of height distribution Sku 3.5±0.4 3.3±0.5

[0214] Root mean square gradient of the surface Sdq 0.43±0.03 0.45±0.03

[0215] Developed area ratio Sdr % 8.0±0.8 8.6±1.0

[0216] Arithmetic mean peak curvature Spc mm-117.4±0.9 12.1±7.5

[0217] Mean dale volume Sdv 104pm356.2±11.4 32.5±6.3

[0218] Areal material ratio Smr(c=0pm) % 0.004±0.0 0.008±0.01

[0219] Inverse areal mat ratio Smc(mc=80%) pm 33.4±0.1 30.6±3.3

[0220] Void volume of valleys Vvv ml / m22.40±0.36 3.66±0.19

[0221] Void volume of the core Vvc ml / m232.5±0.7 27.7±4.1

[0222] Material volume of peaks Vmp ml / m21.61 ±0.15 0.84±0.10

[0223] Material volume of core Vmc ml / m221.3±1.3 24.1 ±3.4

[0224] Void volume of the core

[0225] (mc=20%) Vvc(20 / 80) ml / m220.8±0.8 20.7±2.9

[0226] Material volume of peaks

[0227] (mc=20%) Vmp(20 / 80) ml / m23.59±0.34 2.04±0.20

[0228] Material volume of core

[0229] (mc=20%) Vmc(20 / 80) ml / m219.3±1.5 22.9±3.1 Table 2. Parameters of microbowls

[0230] Diameter of Microbowl Microbowl

[0231] Microbowl Microbowl Microbowl BSCR used glass diameter curvature

[0232] (pB) depth (pm) radius (pm) counts beads (pm) (pm) K(mrrr1) pB-k28-BSCR

[0233] 75 72.0 ±1.1 25.0 ±2.2 36.1 ± 1.3 27.7 ±0.9 18

[0234] 18 pB-k14-BSCR

[0235] 150-210 155.4 ±7.6 57.8 ± 8.5 69.5 ± 6.5 14.4 ±1.2 0

[0236] 0 pB-k11-BSCR

[0237] 250 214.6 ± 12.3 83.2 ±4.3 93.1 ±2.7 10.7 ±0.3 0

[0238] 0

[0239] Table 3. Parameters of microgrooves

[0240] Groove Groove

[0241] Interspace cross- crossMicrogroove Groove Groove BSCR between sectional sectional

[0242] (pG) width (pm) depth (pm) counts grooves (pm) radius curvature

[0243] (pm) K(mm'1) pG-k32-BSCR 1161.6 ±0.7 34.0 ± 0.4 25.6 ± 0.8 31.3 ±0.3 31.9 ±0.311 pG-k17-BSCR5 85.7 ± 5.4 56.1 ±0.5 19.7 ±1.1 58.0 ± 3.5 17.3 ±1.15 pG-k7-BSCR 0 161.3 ±4.833.1 ±3.9 25.7±1.6 145.6 ± 7.56.9 ± 0.3 0

Claims

CLAIMS:

1. A substrate defined as blastocyst motif substrate (BMS) intended for use in cell culture, the substrate comprising a surface intended to be in contact with cells to be cultured, the surface comprising at least one blastocyst motif substrate (BMS) microstructure, wherein said BMS microstructure is characterized in that said BMS microstructure comprises a maximum height within the range of from 5 pm to 100 pm, a maximum width within the range of from 20 pm to 150 pm and a blastocyst scaled curvature range (BSCR) of from 15 to 62 mm-1, wherein the at least one BMS microstructure comprises the BSCR curvature in at least one direction.

2. The blastocyst motif substrate of claim 1 , wherein the at least one BMS microstructure is concave.

3. The blastocyst motif substrate of anyone of the preceding claims, wherein the at least one BMS microstructure comprises a maximum height within the range of from 5 pm to 48 pm; preferably of from 5 pm to 48 pm, more preferably of from 5 pm to 40 pm, even more preferably of from 8 pm to 38 pm; or a range selected from 10 pm to 35 pm or 12 pm to 30 pm.

4. The blastocyst motif substrate of anyone of the preceding claims, wherein the at least one BMS microstructure comprises a maximum width within the range of from 25 pm to 80 pm; preferably of from 29 pm to 79 pm; more preferably 30 pm to 75 pm, even more preferably of from 40 pm to 72 pm; or a range selected from 45 pm to 70 pm or 55 pm to 65 pm.

5. The blastocyst motif substrate of anyone of the preceding claims, wherein the at least one BMS microstructure comprises a BSCR within the range of from 16 to 45 mm-1,preferably of from 18 to 42 mm-1; more preferably of from 20 to 40 mm-1; even more preferably of from 25 to 35 mm-1.

6. The blastocyst motif substrate of anyone of the preceding claims, wherein the blastocyst motif substrate comprises a cell culture area of 300 pm2or more, preferably of 500 pm or more.

7. The blastocyst motif substrate of anyone of the preceding claims, wherein the at least one BMS microstructure is radial symmetric or lateral symmetric; preferably the at least one BMS microstructure has the shape of a bowl or a groove.

8. The blastocyst motif substrate of anyone of the preceding claims, wherein said surface comprises a plurality of BMS microstructures; preferably said plurality of BMS microstructures are arranged on said surface in an orderly or unregular geometric pattern.

9. The blastocyst motif substrate of anyone of the preceding claims, wherein said surface or said at least one BMS microstructure is coated; preferably coated with at least one protein, polymer or extracellular matrix protein component; more preferably the polymer is chosen from poly(ethylene glycol) (PEG), polyvinyl alcohol (PVA), poly-L-lysine (PLL), poly-D- lysine (PDL), polydopamine, chitosan, silk fibroin, and any mixture thereof, and / or the extracellular matrix component is chosen from laminin, collagen, gelatine, entactin, hyaluronic acid, heparin sulphate proteoglycans, Matrigel / Geltrex, and any mixture thereof.

10. The blastocyst motif substrate of anyone of the preceding claims, wherein said surface is arranged at the bottom or at an inner wall of the blastocyst motif substrate; preferably said surface is arranged at the bottom or inner wall facing a volume, cavity or interior of the blastocyst motif substrate.

11. The blastocyst motif substrate of anyone of the preceding claims, wherein the blastocyst motif substrate comprises or consists of a cell culture dish, a cell culture bottle, a cell culture plate, a multi-well cell culture plate, a cell culture bucket and / or a cell culture insert suitable for insertion into one of a cell culture dish, a cell culture bottle, a cell culture plate, a well of a multi-well cell culture plate, a cell culture bucket and / or forms part of an implant or graft.

12. The blastocyst motif substrate of anyone of the preceding claims, wherein the blastocyst motif substrate comprises or consists of a material suitable for cell culture; preferably the material comprises or consists of a plastic polymer, a hydrogel / polymer, a ceramic, or a metal or an alloy thereof; more preferably the plastic polymer / block copolymer is chosen from polystyrene (PS), polypropylene (PP), glycol-modified polyethylene terephthalate (PETG), polyethylene terephthalate (PET), polycarbonate (PC), polycaprolactone (PCL), polylactide (PLA), polydimethylsiloxane (PDMS), polyurethane (Pll), poly(p-dioxanone) (PPDO), poly(pentadecalactone) (PPDL), or any mixture thereof; and / or the hydrogel / polymer is chosen from poly(N-isopropylacrylamide)(PNIPAM), poly(ethylene glycol) (PEG), poly(L-lactic acid-co-glycolic acid) (PLGA), poloxamers (Pluronics), polyvinyl alcohol (PVA), polyacrylamide (PAM), gelatine, collagen, laminin, fibrin, alginate, chitosan, hyaluronic acid, agarose, polydopamine or any mixture thereof, and / or the ceramic is chosen from hydroxyapatite, tricalcium phosphate, calcium phosphate cements, bioactive glass, zirconia, alumina, silica, calcium silicate, magnesium phosphate ceramics, chitosan-ceramic composites, strontium-doped hydroxyapatite, or any mixture thereof, and / or the metal is chosen from gold, silver, platinum, palladium, titanium and titanium alloys, stainless steel, cobalt-chromium alloys, nickel-titanium alloys, tantalum, magnesium alloys, or any mixture thereof.

13. Method of culturing cells, said method comprising the use of a blastocyst motif substrate according to one of claims 1 to 12.

14. The method of claim 13, wherein said method comprises the steps of seeding cells on the blastocyst motif substrate according to one of claims 1 to 12 so that at least some of thecells are in direct or indirect contact with the at least one BMS microstructure and culturing the cells; preferably the cells are cultured on the blastocyst motif substrate for at least 3 hours; more preferably for at least 12 hours; even more preferably for at least 24h.

15. Use of a blastocyst motif substrate according to one of claims 1 to 12 or a method according to claim 13 or 14 in culturing of stem cells; preferably in culturing stem cells to generate naive pluripotent stem cells; more preferably in culturing stem cells to reverse primed stem cells into naive pluripotent stem cells and / or to stabilize such naivety.

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