Improving stem cell differentiation potency using simulated microgravity

Abstract

The present disclosure provides methods for improving the differentiation potency using simulated microgravity. The improvement in differentiation is relative to stem cells that are cultured at 1 g (i.e., at Earth's gravity). In various examples, the improvement is at least 2x better than cultures relative to stem cells that are cultured at 1 g. Also disclosed are methods of differentiating cells cultured by the methods described herein.

Description

IMPROVING STEM CELL DIFFERENTIATION POTENCY USING SIMULATED MICROGRAVITYCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 63 / / 651,916, filed on May 24, 2024, the disclosure of which is hereby incorporated by reference.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML formate and is hereby incorporated by reference in its entirety. Said XML copy, created on May 17, 2025, is named “058636_00797_ST26.xml”, and is 8,158 bytes in size.BACKGROUND OF THE DISCLOSURE

[0003] Stem cells can be used in regenerative medicine, cosmetic and plastic surgery, treatment of degenerative diseases, treatment of cardiovascular diseases, and treatment of neurological diseases. However, millions of cells are required for therapeutics and there are not efficient ways to produce such quantities. Production of specific cell types can be difficult and time consuming to grow without changing their characteristics.

[0004] The capacity of stem cells for self-renewal and differentiation into multiple lineages is tightly regulated by intrinsic genetic programs and extrinsic environmental cues, including biochemical signals and mechanical forces. Mechanical stimuli influence stem cell fate decisions through mechanotransduction pathways, wherein cells convert physical forces into biochemical signals that regulate gene expression, cytoskeletal organization, and lineage commitment. In microgravity conditions — whether during spaceflight or simulated using ground-based systems such as random positioning machines (RPM) or rotating wall vessel bioreactors — mechanotransduction is significantly altered, leading to profound changes in cellular behavior. Understanding how stem cells respond to mechanical unloading is critical not only for optimizing their therapeutic potential in regenerative medicine but also for ensuring their functionality in space-based applications, such as tissue engineering, astronaut health management, and long-duration space missions.

[0005] Among mesenchymal stem cells (MSCs), adipose-derived stem cells (ADSCs) have gained prominence due to their accessibility, high proliferation capacity, and multilineage differentiation potential. Unlike bone marrow-derived MSCs (BM-MSCs), iwhich require invasive harvesting procedures, ADSCs can be obtained from subcutaneous fat via minimally invasive liposuction. Their robust expansion potential and immunomodulatory properties make them ideal candidates for cell-based therapies, including bone and cartilage regeneration, wound healing, and immune modulation. Compared to other stem cell types, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), ADSCs offer distinct advantages for clinical applications. They are easier to isolate, pose a lower risk of teratoma formation, and have strong immunomodulatory capabilities, making them suitable for allogeneic transplantation. While ESCs and iPSCs possess greater pluripotency, their use is limited by ethical concerns, the risk of tumorigenesis, and challenges in controlled differentiation. These factors position ADSCs as a preferred choice for regenerative medicine and therapeutic applications.

[0006] Previous studies on various stem cell types, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), suggest that microgravity can either maintain pluripotency or direct differentiation toward specific lineages. For instance, ESCs and iPSCs exposed to microgravity tend to favor mesodermal and meso-endodermal differentiation, with upregulation of key transcription factors such as Tbx3, Foxa2, and Sox 17, which are associated with cardiogenic and endodermal fates. Similarly, MSCs exhibit lineage-specific differentiation biases under microgravity conditions, often favoring osteogenic differentiation at the expense of adipogenic commitment, or vice versa, depending on culture conditions and exposure duration. These effects are believed to stem from alterations in Wnt, TGF-P, and LIF / STAT3 signaling pathways, as well as changes in cytoskeletal organization and oxidative stress responses. The mechanical environment plays a crucial role in regulating lineage-specific transcription factors, shaping stem cell fate in response to microgravity -induced mechanosensitive changes. Despite these insights, the specific impact of microgravity on ADSC differentiation remains unclear. Given that ADSCs serve as a key source of mesenchymal progenitors, understanding their response to mechanical unloading is particularly relevant for tissue engineering and regenerative medicine, both on Earth and in space. Furthermore, differentiation bias in ADSCs may be influenced by donor variability, microgravity exposure duration, and the specific simulation platform used. While some studies suggest that MSCs retain their undifferentiated state under microgravity, others indicate that prolonged exposure may induce cellular senescence, characterized by increased expression of pl 6, p21, and p53. These findings highlight critical questions regarding the stability of ADSCs in microgravity and their potential applications in regenerative therapies.BRIEF SUMMARY OF THE DISCLOSURE

[0007] In an aspect, the present disclosure provides a method for improving stem cell differentiation in a stem cell population. The improvement in differentiation is relative to stem cells that are cultured at 1 (i.e., at Earth’s gravity). For example, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the stem cell population can differentiate under differentiation conditions following culturing under microgravity (e.g., simulated microgravity). In various examples, the improvement is at least 2x better than cultures relative to stem cells that are cultured at 1 g (i.e., at Earth’s gravity). In various embodiments, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% or more of the cell population can further differentiate under differentiation conditions relative to conventional approaches (i.e., relative to stem cells that are cultured at 1 g (i.e., without microgravity)).

[0008] In various examples, a method for improving stem cell differentiation potency in a stem cell population comprises culturing the cell population under microgravity (e.g., simulated microgravity). The culturing under microgravity (e.g., simulated microgravity) is performed for 48 hours or less, 47 hours or less, 46 hours or less, 45 hours or less, 44 hours or less, 43 hours or less, 42 hours or less, 41 hours or less, 40 hours or less, 39 hours or less, 38 hours or less, 37 hours or less, 36 hours or less, 35 hours or less, 34 hours or less, 33 hours or less, 32 hours or less, 31 hours or less, 30 hours or less, 29 hours or less, 28 hours or less, 27 hours or less, 26 hours or less, 25 hours or less, 24 hours or less, 23 hours or less, 22 hours or less, 21 hours or less, 20 hours or less, 19 hours or less, 18 hours or less, 17 hours or less, 16 hours or less, 15 hours or less, 14 hours or less, 13 hours or less, or about 12 hours. In various examples, the culturing under microgravity (e.g., simulated microgravity) occurs for about 12 hours to about 36 hours. In various examples, the culturing under microgravity (e.g., simulated microgravity) occurs for about 15 hours to about 30 hours. In various examples, the culturing under microgravity occurs for about 20 hours to about 28 hours. In various examples, the culturing under microgravity (e.g., simulated microgravity) occurs for about 24 hours.BRIEF DESCRIPTION OF THE FIGURES

[0009] For a fuller understanding of the nature and objects of the disclosure, reference should be made to the following detailed description taken in conjunction with the accompanying figures.

[0010] FIG. 1 shows the general use of types of stem cells in therapy.

[0011] FIG. 2 shows an outline for stem cell therapy.

[0012] FIG. 3 shows a cartoon for a method of the present disclosure utilizing a random-position machine (RPM).

[0013] FIG 4A shows microscopy images of differentiated stem cells proliferated by a method of the present disclosure at various time points.

[0014] FIG. 4B shows the number of adherent cells at various time points for various cell batches of cells proliferated by a method of the present disclosure.

[0015] FIG. 4C shows the expression change of various cell surface markers at various time points for various cell batches of cells proliferated by a method of the present disclosure.

[0016] FIG. 4D shows the gene expression change of various cell genetic markers at various time points for various cell batches of cells proliferated by a method of the present disclosure.

[0017] FIG. 5A shows microscopy images for adipocyte differentiation from cells proliferated by conventional method and method of the present disclosure at various time points.

[0018] FIG. 5B shows the fold change of number fat cells at various time points for various cell batches differentiated by a method of the present disclosure.

[0019] FIG. 5C shows imaging of adipocyte cells and a distribution of lipid size.

[0020] FIG. 5D shows the number of fat droplets at various time points for various cell batches of cells differentiated by a method of the present disclosure.

[0021] FIG. 5E shows the lipid size at various time points for various cell batches of cells differentiated by a method of the present disclosure.

[0022] FIG. 6A shows microscopy images at different cultivation times for bone differentiation from cells proliferated by conventional method and method of the present disclosure at various time points.

[0023] FIG. 6B shows the fold change of area of mineralization in cells at various time points for various cell batches differentiated by a method of the present disclosure.

[0024] FIG. 6C shows the fold change of area of mineralization in cells at various time points for various cell batches differentiated by a method of the present disclosure.

[0025] FIG. 6D shows microscopy images of bone cells.

[0026] FIG. 6E shows the number of cells with ALP activity at various time points for various cell batches of cells differentiated by a method of the present disclosure._ A >

[0027] FIG. 6F shows intensity of ALP fluorescence signal at various time points for various cell batches of cells differentiated by a method of the present disclosure.

[0028] FIG. 7 shows microscopy imaging of human adipose-derived stem cells (hADSC) culture under different shear force conditions. The cells show different morphology.

[0029] FIG. 8 shows computer simulation shows shear force in microvessel is similar to that of capillary blood flow.

[0030] FIG. 9A shows microscopy images of undifferentiated, adipogenic differentiated cells, and osteogenic differentiated cells from hADSC cultured out of a 2D orbital shaker.

[0031] FIG. 9B shows fold change of gene expression of oct4, sox2, and nanog.

[0032] FIG. 9C shows fold change in differentiation capacity of adipogenic cells(number of fat cells) and osteogenic cells (amount of mineralization).

[0033] FIG. 10 shows microscopy images of cryopreserved cells. Preservation was for one month and adipogenic differentiation was observed. hADSC appears to maintain their “improved potency” after 1 month of cryopreservation.

[0034] FIG. 11 shows a cartoon depicting the effect of gravity and buoyancy on an object.

[0035] FIG. 12 shows a cartoon describing stem cells.

[0036] FIG. 13 shows a picture with cell culture media on an RPM.

[0037] FIG. 14 shows the design of the microvessel used in the cultures, a calculation of the orifice diameter, a picture showing the vessel filled with culture media / liquid, and the liquid weight at various time points.

[0038] FIG. 15 shows images with the differences of cell culture media after six days in wells with a microvessel and without a microvessel; HIFla gene expression; and microscopy images of various cell types after growth and differentiation with and without a microvessel present.

[0039] FIG. 16 shows a cartoon depicting the use of stem cells differentiated by a method of the present disclosure.

[0040] FIG. 17 shows the fluid velocity of fluid in a cell culture flask and the microvessel.

[0041] FIG. 18 shows random imaging of a well filled with cells to count the number of fat cells present.

[0042] FIG. 19 shows images used for quantitative analysis of alizarin red s-stained cells.

[0043] FIG. 20 shows the % change in number of cells and effects of Simulated Microgravity on cell proliferation, death, and DNA damage in relation to the data presented in FIG. 4B.

[0044] FIG. 21 shows images to substantiate that the effects of Simulated Microgravity on ADSC is due to reduction of lamin A / C.

[0045] FIG. 22 shows effects of Simulated Microgravity on ADSC Proliferation, Morphology, and Stem Cell Marker Expression. (Left panel) Quantification of the normalized cell number at each time point. (D-F) Fold change in stem cell marker expression over time for (Panel second from left) CD44, (Panel third from left) CD90, and (Rightmost panel) CD105. All data are normalized to ADSCs cultured under normal gravity (1g) at the corresponding time point. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test for pairwise comparisons between time points. Different dot colors represent individual cell batches, and lines indicate paired data from the same experimental run, tracking changes in each batch over time. Statistical significance is denoted as follows: ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Experiments were performed using three different donors, each with four independent replicates.

[0046] FIG. 23 A shows transcriptomic Analysis of ADSCs Under Simulated Microgravity. Hierarchical clustering heatmap of differentially expressed genes in ADSCs cultured under simulated microgravity (sug) and static conditions at day 1 and day 4. Red represents upregulated genes, while green represents downregulated genes.

[0047] FIG. 23B shows transcriptomic Analysis of ADSCs Under Simulated Microgravity. Principal Component Analysis (PCA) of gene expression profiles, showing distinct clustering based on culture conditions and time points.

[0048] FIG. 23C shows transcriptomic Analysis of ADSCs Under Simulated Microgravity. (C) Venn diagrams illustrating the overlap of upregulated and downregulated genes with fold change (FC) > 2 and false discovery rate (FDR) < 0.05 across different comparisons. Gene Ontology (GO) enrichment analysis of differentially expressed genes for day 1 (sug vs. static), day 4 (sug vs. static), and day 4 vs. day 1 under simulated microgravity.

[0049] FIG. 23D shows transcriptomic Analysis of ADSCs Under Simulated Microgravity. Venn diagrams illustrating the overlap of upregulated and downregulated genes with fold change (FC) > 2 and false discovery rate (FDR) < 0.05 across differentcomparisons. Gene Ontology (GO) enrichment analysis of differentially expressed genes for day 1 (sug vs. static) under simulated microgravity.

[0050] FIG. 23E shows transcriptomic Analysis of ADSCs Under Simulated Microgravity. Venn diagrams illustrating the overlap of upregulated and downregulated genes with fold change (FC) > 2 and false discovery rate (FDR) < 0.05 across different comparisons. Gene Ontology (GO) enrichment analysis of differentially expressed genes for day 4 (sug vs. static) under simulated microgravity.

[0051] FIG. 23F shows transcriptomic Analysis of ADSCs Under Simulated Microgravity. Venn diagrams illustrating the overlap of upregulated and downregulated genes with fold change (FC) > 2 and false discovery rate (FDR) < 0.05 across different comparisons. Gene Ontology (GO) enrichment analysis of differentially expressed genes for day 4 vs. day 1 under simulated microgravity.DETAILED DESCRIPTION OF THE DISCLOSURE

[0052] Although claimed subject matter will be described in terms of certain embodiments, other embodiments, including embodiments that do not provide all of the benefits and features set forth herein, are also within the scope of this disclosure. Various structural, logical, and process step changes may be made without departing from the scope of the disclosure.

[0053] As used herein, unless otherwise indicated, “about”, “substantially”, or “the like”, when used in connection with a measurable variable (such as, for example, a parameter, an amount, a temporal duration, or the like) or a list of alternatives, is meant to encompass variations of and from the specified value including, but not limited to, those within experimental error (which can be determined by, e.g., a given data set, an art accepted standard, etc. and / or with, e.g., a given confidence interval (e.g. 90%, 95%, or more confidence interval from the mean), such as, for example, variations of + / - 10% or less, + / -5% or less, + / -1% or less, and + / -0.1% or less of and from the specified value), insofar such variations in a variable and / or variations in the alternatives are appropriate to perform in the instant disclosure. As used herein, the term “about” may mean that the amount or value in question is the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, compositions, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error, or the like, or other factors known to those of skill in the artsuch that equivalent results or effects are obtained. In general, an amount, size, composition, parameter, or other quantity or characteristic, or alternative is “about” or “the like,” whether or not expressly stated to be such. It is understood that where “about,” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0054] Ranges of values are disclosed herein. The ranges set out a lower limit value and an upper limit value. Unless otherwise stated, the ranges include the lower limit value, the upper limit value, and all values between the lower limit value and the upper limit value, including, but not limited to, all values to the magnitude of the smallest value (either the lower limit value or the upper limit value) of a range. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “0.1% to 5%” should be interpreted to include not only the explicitly recited values of 0.1% to 5%, but also, unless otherwise stated, include individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5% to 1.1%; 0.5% to 2.4%; 0.5% to 3.2%, and 0.5% to 4.4%, and other possible sub-ranges) within the indicated range. It is also understood (as presented above) that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about, it will be understood that the particular value forms a further disclosure. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0055] The present disclosure provides methods for improving the differentiation potency using simulated microgravity. Also disclosed are methods of differentiating cells cultured by the methods described herein.

[0056] The term “stem cells” as used herein refers to undifferentiated cells prior to differentiation into individual cells constituting tissues, which have the ability to differentiate into specific cells under specific stimulation (e.g., environment). Unlike differentiated cells whose cell division has stopped, the stem cells retain the capability of self-renewal through cell division and have the plasticity of differentiation to differentiate into various cells under different stimuli.

[0057] The term “microgravity” as used herein refers to zero gravity or undetectably small gravity. Specifically, it refers to an environment that experiences gravity of a range of l x IO'2to l x IO'6. The term “microgravity” may also be expressed as “weightlessness.” “Microgravity” may include “simulated microgravity” (sug, spg, suG, or spG) For example, “simulated microgravity” is when microgravity is simulated via the use of a machine or instrument, such as, for example, a random position machine (RPM). Under “simulated microgravity,” the gravity experienced may vary at any given time and is not constant. Thus, the gravity experienced during simulated microgravity may include anywhere from actual microgravity, anything less than 1 g, and gravity greater than 1 g, but the resulting average gravity is less than 1 g. “Average gravity” may be referred to as “perceived gravity” or “effective gravity.”

[0058] In an aspect, the present disclosure provides a method for improving stem cell differentiation in a stem cell population. The improvement in differentiation is relative to stem cells that are cultured at 1 g (i.e., at Earth’s gravity). For example, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the stem cell population can differentiate under differentiation conditions following culturing under microgravity (e.g., simulated microgravity). In various examples, the improvement is at least 2x better than cultures relative to stem cells that are cultured at 1 g (i.e., at Earth’s gravity). In various embodiments, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% or more of the cell population can further differentiate under differentiation conditions relative to conventional approaches (i.e., relative to stem cells that are cultured at 1 g (i.e., without microgravity (e.g., simulated microgravity))).

[0059] In various examples, stem cells are exposed to gravity where the average gravity the stem cells experience is less than 1 g. For example, average gravity is 0 to 0.9 g, including every 0.01 value and range therebetween. In various examples, average gravity is 0.49g.

[0060] In various examples, a method for improving stem cell differentiation potency in a stem cell population comprises culturing the cell population under microgravity (e.g., simulated microgravity). The culturing under microgravity (e.g., simulated microgravity) is performed for 48 hours or less, 47 hours or less, 46 hours or less, 45 hours or less, 44 hours or less, 43 hours or less, 42 hours or less, 41 hours or less, 40 hours or less, 39 hours or less, 38 hours or less, 37 hours or less, 36 hours or less, 35 hours or less, 34 hours or less, 33 hours or less, 32 hours or less, 31 hours or less, 30 hours or less, 29 hours or less, 28 hours or less, 27hours or less, 26 hours or less, 25 hours or less, 24 hours or less, 23 hours or less, 22 hours or less, 21 hours or less, 20 hours or less, 19 hours or less, 18 hours or less, 17 hours or less, 16 hours or less, 15 hours or less, 14 hours or less, 13 hours or less, or about 12 hours. In various examples, the culturing under microgravity (e.g., simulated microgravity) occurs for about 12 hours to about 36 hours. In various examples, the culturing under microgravity (e.g., simulated microgravity) occurs for about 15 hours to about 30 hours. In various examples, the culturing under microgravity (e.g., simulated microgravity) occurs for about 20 hours to about 28 hours. In various examples, the culturing under microgravity (e.g., simulated microgravity) occurs for about 24 hours.

[0061] During culture under microgravity (e.g., simulated microgravity), various vessels may be used. For example, the vessel may be a convention cell culture flask. In various other examples, the stem cells are cultured in a microvessel. Examples of suitable microvessels are described in El Gindi etal., Engineered Microvessel for Cell Culture in Simulated Microgravity, Int. J. Mol. Sci., 2021, 22(12), 6331, the disclosure of which is incorporated herein in its entirety. In various examples, the microvessel is disposed in a multiwelled plate. In various examples, the microvessel is made from polydimethylsiloxane (PDMS).

[0062] In various examples, the microgravity is simulated using a random position machine (RPM). An RPM is capable of offsetting gravity by rotating continuously while changing directions randomly or according to a preset pattern. In various examples, the culturing is performed on an RPM, where the RPM is moving the culturing vessel (e.g., multiwelled plate comprising one or more microvessels) at an average velocity of 45 to 75 deg / s, including all 0.1 deg / s values and ranges therebetween. In various examples, the average velocity is 60 deg / s. In various examples, the RPM moves in a random direction.

[0063] Various stem cells may be cultured under microgravity (e.g., simulated microgravity). In various examples, the stem cells, prior to differentiation, are human adipose-derived stem cells (hADSC). In various other examples, suitable examples of stem cells include, but are not limited to, bone marrow derived stem cells, stem cells from other organs (e.g., cardiac stem cells, dental root stem cells, gut stem cells, and the like) and induced pluripotent stem cells (iPSC). Other examples of cells include, but are not limited to, perinatal stem cells; human embryonic stem cells, human adult mesenchymal stem cells (such as, for example, ADSC, bone marrow derived stem cells, and stem cells from other adult tissues), Human hematopoietic stem cells (covers adult blood derived stem cells), and iPSCs.

[0064] Various culture media can be used for culturing under microgravity (e.g., simulated microgravity). Various examples of culture media include, but are not limited to, Gibco MesenPRO RS™ media. Other culture media are known in the art and are suitable. Any other media known for culturing other forms of stem cells and IPSC cells are suitable for use in the present disclosure.

[0065] Prior to culturing the stem cell population under microgravity (e.g., simulated microgravity), various methods may be used to grow the stem cell population to a desired confluency. For example, the stem cells may be maintained and passaged to reach about 80% confluency. In various examples, the stem cell population is cultured for 24 hours prior to culturing under microgravity to allow for a desirable amount of cell adherence.

[0066] Following culturing under microgravity (e.g., simulated microgravity), the stem cell population may be cryopreserved for long term storage or differentiated. In various examples, following long term storage, the stem cells may be thawed and then differentiated with little to no change in differentiation ability relative to a stem cell population cultured under microgravity that has not been stored. The stem cell population cultured under microgravity may be stored in a medium comprising a cell culture medium, fetal bovine serum (FBS), and dimethylsulfoxide (DMSO) further stored in a cryogenic vial. The cryogenic vial may be placed in an isopropanol freezing container and then placed in a -80 °C freezer.

[0067] Following culturing the stem cell population under microgravity (e.g., simulated microgravity), the stem cell population may be differentiated. As described herein, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% of the stem cell population can differentiate under differentiation conditions following culturing under microgravity (e.g., simulated microgravity). For example, the amount of time a stem cell population is in contact with a specific differentiation medium can affect the resulting differentiation. For example, adipocytes can be formed after contacting the stem cell population with adipogenic differentiation medium for about 14 days, whereas osteocytes can be formed after contacting the stem cell population with osteogenic differentiation medium for about 21 days. The differentiation medium can be replaced about every three days.

[0068] Stem Cell Differentiation into Adipocytes and Osteocytes may be achieved by: Human adipose-derived stem cells (hADSCs) exposed to simulated microgravity (spG) were washed twice with phosphate-buffered saline (PBS) and detached using an enzymatic dissociation method (TrypLE, Gibo, Thermo Fischer Scientific). A total of about 5 * 104cellswere seeded per well in a 24-well plate (Nunc, Thermo Fischer Scientific) and maintained in specific differentiation media. For adipogenic differentiation, cells were cultured in about 500 pl of StemPro™ Adipogenesis Differentiation Medium (Thermo Fisher Scientific, Dreieich, Germany) for about 14 days, while osteogenic differentiation was induced using about 500 pl StemPro™ Osteogenesis Differentiation Medium (Thermo Fisher Scientific) for about 21 days. The differentiation media were completely replaced every 3 days to ensure optimal differentiation conditions.

[0069] Quantitative Analysis of Adipogenic Differentiation may be achieved by: After adipogenic differentiation, cells were washed with PBS and fixed with 4% paraformaldehyde for about 10 minutes at room temperature. Subsequently, cells were washed again with PBS and stained for intracellular lipid droplets using Nile Red dye (Ex / Em = 552 / 636 nm) and DAPI (1 : 10,000 dilution) for about 1 hour at room temperature.Following staining, cells were washed with PBS and imaged using a Lionheart FX automated microscope (BioTek, USA) equipped with a 10x objective. The blue channel was used to visualize cell nuclei, while the green channel was used to detect lipid droplets. Differentiated cells were manually counted by identifying cells containing lipid droplets. The obtained data were normalized to adipocytes differentiated under 1g conditions. Additionally, images were analyzed for lipid droplet count and lipid size using the imfindcircles function in MATLAB. These data were normalized to osteocytes differentiated at 1g. For both analyses, images from four positions per sample were acquired and quantified.

[0070] Quantitative Analysis of Osteogenic Differentiation may be achieved by: After osteogenic induction, cells were washed with PBS and stained with 2% Alizarin Red S solution and DAPI (1 : 10,000 dilution) for about 5 minutes. The cells were then rinsed with PBS and imaged using a Lionheart FX automated microscope (BioTek, USA) with a 10x objective to visualize calcium deposits. The area of deposited calcium per image field was quantified using ImageJ (NIH) and normalized to the cell number, as indicated by DAPI staining. To quantify Alizarin Red S staining, 10% cetylpyridinium chloride (Sigma- Aldrich) was added, and cells were incubated for 20 minutes to elute the stain. The supernatants were then collected, and the absorbance of the eluted stain was measured at 570 nm using a plate reader (BioTek Hl). The obtained data were normalized to the cell number per well, as determined by DAPI staining.

[0071] The following Statements provide various examples of the present disclosure. They are not intended to be limiting in any way.Statement 1. A method for improving stem cell differentiation potency in a stem cell population comprising culturing the stem cell population under microgravity for about 12 hours to about 36 hours, wherein at least 70% of the stem cell population can further differentiate under differentiation conditions. In various examples, the microgravity is simulated microgravity. The average gravity may be greater than 0 g but less than 1 g (e.g., 0.49 g).Statement 2. A method according to Statement 1, wherein the stem cell population is cultured in a welled plate comprising a microvessel.Statement 3. A method according to Statement 2, wherein the microvessel is made from polydimethylsiloxane.Statement 4. A method according to any one of the preceding Statements, wherein the microgravity occurs from culturing the stem cell population on a random position machine.Statement 5. A method according to Statement 4, wherein random position machine moves at an average velocity of 45 to 75 deg / s, including all 0.1 deg / s values and ranges therebetween (e.g., 60 deg / s).Statement 6. A method according to Statement 4 or Statement 5, wherein the random position machine moves in a random direction (e.g., one or more random directions).Statement 7. A method according to any one of the preceding Statements, wherein the stem cells are human adipose-derived stem cells, bone marrow-derived stem cells, or induced pluripotent stem cells, or the like. Other examples of cells include, but are not limited to, perinatal stem cells; human embryonic stem cells, human adult mesenchymal stem cells (such as, for example, ADSC, bone marrow derived stem cells, and stem cells from other adult tisuses), Human hematopoietic stem cells (covers adult blood derived stem cells), and iPSCs.Statement 8. A method according to any one of the preceding Statements, wherein at least 80% of the stem cell population can further differentiate under differentiation conditions.Statement 9. A method according to any one of the preceding Statements, wherein at least 85% of the stem cell population can further differentiate under differentiation conditions.Statement 10. A method according to any one of the preceding Statements, wherein at least 90% of the stem cell population can further differentiate under differentiation conditions.Statement 11. A method according to any one of the preceding Statements, wherein the stem cell population was cultured for about 24 hours to allow for cell adherence prior to culturing under microgravity.Statement 12. A method according to any one of the preceding Statements, wherein the stem cells are cultured under microgravity for about 15 hours to about 30 hours.Statement 13. A method according to Statement 12, wherein the stem cells are cultured under microgravity for about 20 hours to about 28 hours.Statement 14. A method according to Statement 13, wherein the stem cells are cultured under microgravity for about 24 hours.Statement 15. A method for differentiating stem cells with an improved differentiation potency, comprising contacting the stem cells that have been cultured under microgravity of any one of the preceding claims with a differentiation medium for a period of time such that stem cells are differentiated to the desired cell type.Statement 16. A method according to Statement 15, wherein the differentiation medium is exchanged at least every three days.Statement 17. A method according to Statement 15 or Statement 16, wherein the stem cells are differentiated for at least 14 days.Statement 18. A method according to Statement 17, wherein the stem cells are differentiated for at least 21 days.

[0072] The steps of the method described in the various embodiments and examples disclosed herein are sufficient to carry out the methods of the present disclosure. Thus, in an embodiment, the method consists essentially of a combination of the steps of the methods disclosed herein. In another embodiment, the method consists of such steps.

[0073] The following examples are presented to illustrate the present disclosure. They are not intended to be limiting in any way.EXAMPLE 1

[0074] This example provides a description of methods of the present disclosure.

[0075] The self-renewal capacity and ability to be differentiated into various specialized cell types make stem cells an optimum source of cells for tissue engineering and regenerative medicine strategies. Decades of research have shown that effective differentiation of stem cells into specialized cells can be enhanced through appropriate use of> 1 A >various extrinsic factors that are both physical and chemical in nature. Here, described herein is that upon exposure to simulated microgravity, the differentiation potential of adipose derived stem cells (ADSC) is augmented. Microgravity, real or simulated, have been known to affect cellular behavior. ADSCs were thus cultured in custom engineered microvessels and placed on a random positioning machine for a duration and subsequently treated with adipogenic and osteogenic factors for 2 weeks. Random positioning machines simulate microgravity by canceling out gravitational vectors. After treatment with simulated microgravity, cells were observed and quantified for lipid droplet formation and alkaline phosphotase to determine adipocyte and osteoblast differentiation respectively. High- throughput sequencing and PCR to was performed further substantiate these findings through sternness markers.

[0076] Maintaining and culture of adipose-derived stem cells. Human primary adipose-derived stem cells (hADSCs) from three different batches were purchased from Thermo Fisher Scientific, Dreieich, Germany (Cat Nr,). The cells were thawed and maintained in Gibco MesenPRO RS™ medium (Cat Nr,), prepared according to the manufacturer's protocols. To maintain the cells, the media were replaced every three days, and the cells were passaged using TrypLE (Cat Nr.) once they reached 80% confluency.

[0077] Preparing cell culture for culture on random positioning machine. Human adipose-derived stem cells (hADSCs) for culture under simulated microgravity (spG) using a random positioning machine were used up to the 8th passage after subculture upon purchase. 5 x 10A4 cells were seeded and cultured in 4-well plates (Nunc, Thermo Fisher Scientific, Dreieich, Germany) using Gibco MesenPRO RS™ cell culture medium for 24 hours to allow for cell adherence. Afterwards, the cell culture medium was removed, and biocompatible microvessels were placed onto the well, as previously described. Fresh Gibco MesenPRO RS™ cell culture medium was then added to the well. The cells were subsequently cultured for 1, 4, and 7 days at 1g or spG conditions in an incubator under standard cell culture conditions.

[0078] Setting for Random Positioning Machine. All experiments using simulated microgravity (spG) were conducted on a desktop Random Positioning Machine (RPM) (Airbus Defence and Space Netherlands B.V., Leiden, The Netherlands). The RPM operated in four modes: (1) constant speed with an average velocity of 60 deg / s and random direction, (2) random speed with an average velocity of 60 deg / s and constant direction, (3) random speed with an average velocity of 60 deg / s and random direction, and (4) a 0g preset from the manufacturer. To maintain an average velocity of 60 deg / s, the minimum and maximumvelocities were set at 45 and 75 deg / s, respectively. In addition to the RPM machine, a 2D orbital shaker was used to culture cells at a velocity of 60 deg / s. To perform cell experiments, prepared four-well cell culture plates were placed at the center of the rotation, as previously described. Unless otherwise stated, all experiments were conducted using random speed with an average velocity of 60 deg / s and random direction (setting nr. 3), in an incubator under standard cell culture conditions.

[0079] Cell imaging and quantitative analysis of cell proliferation and viability.Cell imaging was conducted using a Lionheart FX automated microscope (BioTek, USA) with a 10x objective in bright-field mode. For cell proliferation and viability analysis, flow cytometry was employed. To assess these parameters, cells were detached from tissue culture plastic by incubating them with TrypLE™ Express (Thermo Fisher Scientific, Dreieich, Germany) for 5 minutes under standard cell culture conditions. Cells were stained with DRAQ7 (dilution 1 : 1000 in PBS), a cell-impermeable dye used to indicate dead cells, for 15 minutes on ice. Subsequently, an Attune NxT Flow Cytometer equipped with an autosampler (Thermo Fisher Scientific, USA) was used to analyze the cells. The percentage of dead cells was determined by counting the number of cells positive for DRAQ7. Cell proliferation analysis involved normalizing the cell count obtained from the flow cytometer to the initial seeded cell number (5 x 104cells). Experiments were performed at least in triplicate for each cell batch.

[0080] Quantitative analysis of cell surface markers. To investigate the expression of cell surface markers, cells were detached from the culture well plate by incubating them with TrypLE™ Express (Thermo Fisher Scientific, Dreieich, Germany) for 5 minutes under standard cell culture conditions. Subsequently, the cells were stained with mouse anti-human antibodies against CD44, CD90, and CD105 on ice for 30 minutes. The antibodies were diluted in PBS at a ratio of 1 :250. All antibodies used in this study were purchased from Biolegend, San Diego, USA. The stained cells were analyzed using an Attune NxT Flow Cytometer equipped with an autosampler (Thermo Fisher Scientific, CA, USA). Compensation settings were applied prior to running the analysis. FlowJo software (Becton, Dickinson and Company, NJ, USA) was used for data analysis, and the geometric mean fluorescence intensity (gMFI) of the cell surface markers was determined. Experiments were performed in at least triplicate for each cell batch.

[0081] Stem cell differentiation into adipocytes and osteocytes. Human adipose- derived stem cells (hADSCs) exposed to simulated microgravity (spG) were washed twice with PBS. The cells were detached and 5 x 104were seeded in a 24-well plate. The cells werecultured with commercial adipogenic (StemPro™ adipogenesis differentiation, Cat Nr.) or osteogenic (StemPro™ osteogenesis differentiation, Cat. Nr.) differentiation media purchased from Thermo Fisher Scientific, Dreieich, Germany. The cells were cultured in differentiation media for 14 days for adipogenesis or 21 days for osteogenesis, with a complete exchange of differentiation media every 3 days.

[0082] Quantitative analysis of osteogenic differentiation. After 21 days of osteogenic differentiation, the cells were washed with PBS and stained for Alkaline Phosphatase (ALP) and Alizarin Red S. For staining, the ALP live stain solution was prepared by diluting the 500X stock solution in cell culture media. The cells were cultured with the ALP staining solution for 30 minutes under standard cell culture conditions. Afterwards, the cells were washed twice with PBS and further stained with a 2% Alizarin Red S solution and DAPI (1 : 10000 dilution) for 5 minutes. The cells were then washed with PBS and imaged under a Lionheart FX automated microscope (BioTek, USA) with a 10x objective to visualize the cell nuclei (blue channel), ALP (green channel), and calcium deposits (red channel). The number of cells with and without ALP activity was counted, and mean fluorescence intensities were analyzed by cell segmentation using Biotek microscope software. The obtained data were normalized to osteocytes differentiated at 1g. Additionally, the area of deposited calcium (Alizarin Red S staining, red color) per image field was quantified using ImageJ (NIH) by converting the images to grayscale, followed by thresholding using the Otsu method. The Alizarin Red S staining area was quantified and normalized to the cell number (indicated by DAPI count). Imaging of the samples was performed within 90 minutes after staining. To further quantify the Alizarin Red staining, 10% cetylpyridinium chloride (Sigma Aldrich) was added, and the cells were incubated for 20 minutes to elute the Alizarin Red S stain. Afterwards, the supernatants were collected and quantified by measuring the absorbance of the eluted stain at 570 nm using a plate reader (Biotek Hl). The obtained absorbance data were normalized to the number of cells per well, as determined by the DAPI count. For both analyses, images of 4 positions per sample were taken and quantified.

[0083] Quantitative analysis of adipogenic differentiation. After 14 days of adipogenic differentiation, the cells were washed with PBS and fixed with 4% paraformaldehyde for 10 minutes at room temperature. Subsequently, the cells were washed with PBS and stained for intracellular lipid droplets using Nile Red dye (Ex / Em = 552 / 636 nm) and DAPI (1 : 10000 dilution) for 1 hour at room temperature. Afterwards, the cells were washed with PBS and imaged under a Lionheart FX automated microscope (BioTek, USA)with a 10x objective to visualize the cell nuclei (blue channel) and fat droplets (green channel). The number of differentiated cells was manually counted by identifying cells containing lipid droplets. The obtained data were normalized to adipocytes differentiated at 1g. Additionally, the images were analyzed for the number of lipid droplets and lipid size using the imfmdcircles function in MATLAB software. The obtained data were normalized to osteocytes differentiated at 1g. For both analyses, images of 4 positions per sample were taken and quantified.

[0084] RNA Isolation and Gene Expression Analysis. RNA was extracted using TRIzol (Invitrogen, Thermo Fisher Scientific, Inc., Dreieich, Germany), followed by chloroform extraction (Sigma- Aldrich, Schnelldorf, Germany) using the manufacturer’s protocol. The RNA concentration and purity (the ratio of absorbance at 260 nm and 280 nm) were quantified using Nanodrop (Thermo Fisher Scientific, Inc., Dreieich, Germany). RNA was subsequently converted into complementary DNA (cDNA) using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Thermo Fisher Scientific, Inc., Dreieich, Germany). The primers used in this study were synthesized from GenScript Biotech, Piscataway, NJ, USA. qPCR was performed using the SYBR Green PCR Master Mix (Applied Biosystems, Thermo Fisher Scientific, Inc., Dreieich, Germany). The primer sequences used are listed in Table 1. The qPCR procedure was set as follows: denaturation for 5 min at 95 °C; 45 cycles of denaturation (95 °C, 15 s), annealing under primer-specific conditions (30 s), and target gene-specific extension (30 s at 72 °C). Fluorescence signals were measured for 20 s at 72 °C. To confirm the specificity of the PCR products, a melting curve analysis was performed at the end of each run. The RPS26 gene was used as a reference gene. The relative expression levels were calculated using the 2-AACTmethod. Experiments were performed with at least in three independent replicates per cell batch.

[0085] Table 1 : Primers used in this study

[0086] Cryopreservation of sug-exposed stem cells. Human adipose-derived stem cells (hADSCs) cultured on an RPM machine were harvested on days 1 and 4 for cryopreservation. The cell culture cryopreservation medium consists of 50% MesenPRO RS™ medium, 40% FBS, and 10% DMSO. 5 x 105cells were added to 1 mL of the prepared cry opreservation medium in a cryogenic vial. Controlled-rate freezing was performed by placing the cryogenic vials in an isopropanol freezing container and then into a -80°C freezer to cool slowly overnight. Afterwards, the cells were kept in a liquid nitrogen tank for one month prior to testing for gene expression related to sternness and differentiation into adipocytes or osteocytes.

[0087] Data and Statistical Analysis. Statistical significance was determined by two- way ANOVA followed by Tukey’s post hoc test using Prism 10 (GraphPad Software Inc., San Diego, CA, USA) and the level of significance was set to < 0.05. Unless otherwise stated, all experiments were performed with at least three replicates and data are represented as mean ± standard deviation (SD).EXAMPLE 2

[0088] This example provides a description of methods of the present disclosure.

[0089] This study investigates the effects of simulated microgravity on ADSCs by examining cell proliferation, morphology, and stem cell marker expression to evaluate their mechanosensitive responses. Additionally, adipogenic and osteogenic differentiation assays were performed to assess how microgravity influences ADSC differentiation potential.Understanding how ADSCs adapt to microgravity is essential for their potential application in space-based regenerative medicine. The findings from this study will provide valuable insights into the broader impact of microgravity on stem cell biology, with implications for both space and terrestrial medicine. Moreover, these insights may help refine strategies for optimizing ADSC-based therapies, ensuring their efficacy in regenerative treatments under different mechanical conditions.

[0090] Materials and Methods

[0091] Cell Culture. Human adipose-derived stem cells (hADSCs) from three different donors were purchased from Thermo Fisher Scientificlnc, Dreieich, Germany. Cells were cultured in MesenPRO RS™ Medium (Thermo Fisher Scientificlnc, Dreieich, Germany.) according to the manufacturer’s instructions. Cells were maintained at 3 7°C, 95% humidity with 5% CO2. Cells were expanded and used for experiments up to passage 8. i a >

[0092] Setting Up the Random Positioning Machine. All experiments involving simulated microgravity (spG) were conducted using a desktop Random Positioning Machine (RPM) (Airbus Defence and Space Netherlands B.V., Leiden, The Netherlands). The RPM was operated in 3D random mode, utilizing random motion and direction while maintaining an average velocity of 607s. Four-well cell culture plates were positioned at the center of rotation, as previously described. All RPM experiments were performed inside an incubator under standard cell culture conditions.

[0093] Cell imaging and quantitative analysis of cell proliferation and viability.Cell imaging was performed using a Lionheart FX automated microscope (BioTek, USA) with a 10x objective in bright-field mode. Cell proliferation and viability were assessed via flow cytometry. To evaluate these parameters, cells were detached from tissue culture plastic by incubating them with TrypLE™ Express (Thermo Fisher Scientific, Dreieich, Germany) for 5 minutes under standard cell culture conditions. Detached cells were stained with DRAQ7 (1 : 1000 dilution in PBS), a cell-impermeable dye used to identify dead cells, and incubated for 15 minutes on ice. Subsequently, analysis was conducted using an Attune NxT Flow Cytometer equipped with an autosampler (Thermo Fisher Scientific, USA). The percentage of dead cells was determined based on the number of DRAQ7-positive cells. For proliferation analysis, the total cell count obtained from the flow cytometer was normalized to the initial seeding density (5 * 104cells). The experiment was performed with four independent replicates per donor.

[0094] Quantitative analysis of hADSC through the expression of cell surface markers and intracellular transcription factor expression. To quantify cell activation, cells were detached from tissue culture plastic using TrypLE™ Cell Detachment Solution (Thermo Fisher Scientific) and stained with the antibodies (diluted 1 :500 in PBS) from Table 2 for 30 minutes on ice. All antibodies were obtained from BioLegend (San Diego, CA, USA). Cell surface marker expression was quantified by measuring the geometric mean fluorescence intensity (gMFI) using an Attune NxT Flow Cytometer equipped with an autosampler (Thermo Fisher Scientific, Waltham, MA, USA). The experiment was performed with four independent replicates per donor.

[0095] To analyze the expression of transcription factors OCT4, SOX2, and NANOG, cells were detached from tissue culture plastic using TrypLE™ Cell Detachment Solution (Thermo Fisher Scientific), fixed with 4% paraformaldehyde (PF A), permeabilized with 0.1% Triton X-100, and blocked with 1% bovine serum albumin (BSA). Cells were then stained with the antibodies in Table 2 for 30 minutes on ice. After staining, cells were washedwith PBS, and the fluorescence intensity of the transcription factors was quantified using an Attune NxT Flow Cytometer equipped with an autosampler (Thermo Fisher Scientific, Waltham, MA, USA). The experiment was conducted with four independent replicates per donor.

[0096] Table 2. Antibodies used.

[0097] Gene expression analysis. Gene expression analysis was performed using an established protocol. Briefly, RNA was extracted using TRIzol (Invitrogen, USA), and its concentration and 260 / 280 nm absorbance ratio were measured using a NanoDrop Spectrophotometer (Thermo Fisher Scientific, USA). The extracted RNA was then converted into complementary DNA (cDNA) using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Primers were synthesized by Bioneer (Republic of Korea). Quantitative polymerase chain reaction (qPCR) was performed using the SYBR Green PCR Master Mix (Applied Biosystems, USA) under the following thermal cycling conditions: an initial denaturation step at 95 °C for 5 minutes, followed by 45 cycles of denaturation at 95 °C for 15 seconds, annealing under primer-specific conditions for 30 seconds, and extension at 72 °C for 30 seconds. Fluorescence signals were measured for 20 seconds at 72 °C. The RPS26 gene was used as a reference gene, and all primer sequences along with their accession numbers are provided in Supplementary Table 2. To ensure the specificity of the PCR products, a melting curve analysis was conducted at the end of eachrun. Both cDNA synthesis and qPCR were performed using a Stratagene Mx3005P system (Agilent Technologies, CA, USA). Relative gene expression levels were calculated using the 2 AACT method Experiments were conducted in four replicates per donor.

[0098] RNA Sequencing and Analysis. RNA sequencing was performed using the Oxford Nanopore cDNA Sequencing Kit and 24 Barcoding Kit, processed through Epi2me with the wf-transcriptomes workflow (Oxford Nanopore Technologies, Oxford, UK), following the manufacturer’s protocol. Briefly, total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA) and purified with the RNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA libraries were prepared using the NEBNext Poly(A) mRNA Magnetic Isolation Module, dual-indexed, and barcoded with the Oxford Nanopore 24 Barcoding Kit, according to the manufacturer's instructions. Library concentration, size distribution, and quality were assessed using a Qubit 4 Fluorometer (Thermo Fisher, Waltham, MA, USA) and a 4200 TapeStation (Agilent, Santa Clara, CA, USA). Libraries were pooled, normalized, and quantified before sequencing on a PromethlON 2 platform (Oxford Nanopore Technologies, Oxford, UK). RNA-Seq was performed in triplicate. Raw FASTQ reads were processed using Epi2me with the wf-transcriptomes workflow (Oxford Nanopore Technologies, Oxford, UK) under default settings. The reads were aligned to the human reference genome GRCh38.p4, and gene count data were merged using NASQAR and analyzed with iDEP 2.0.Differentially expressed genes (DEGs) were identified using DESeq2, with a false discovery rate (FDR) threshold of < 0.05 and a fold change (FC) > 1.5. Biological pathways were predicted using Generally Applicable Gene-set Enrichment for Pathway Analysis (GAGE).

[0099] Stem Cell Differentiation into Adipocytes and Osteocytes. Human adipose- derived stem cells (hADSCs) exposed to simulated microgravity (spG) were washed twice with phosphate-buffered saline (PBS) and detached using an appropriate enzymatic or non- enzymatic dissociation method. A total of 5 x io4cells were seeded per well in a 24-well plate and maintained in specific differentiation media. For adipogenic differentiation, cells were cultured in StemPro™ Adipogenesis Differentiation Medium (Thermo Fisher Scientific, Dreieich, Germany) for 14 days, while osteogenic differentiation was induced using StemPro™ Osteogenesis Differentiation Medium (Thermo Fisher Scientific) for 21 days. The differentiation media were completely replaced every 3 days to ensure optimal differentiation conditions.

[0100] Quantitative Analysis of Adipogenic Differentiation. After adipogenic differentiation, cells were washed with PBS and fixed with 4% paraformaldehyde for 10minutes at room temperature. Subsequently, cells were washed again with PBS and stained for intracellular lipid droplets using Nile Red dye (Ex / Em = 552 / 636 nm) and DAPI (1 : 10,000 dilution) for 1 hour at room temperature. Following staining, cells were washed with PBS and imaged using a Lionheart FX automated microscope (BioTek, USA) equipped with a 10x objective. The blue channel was used to visualize cell nuclei, while the green channel was used to detect lipid droplets. Differentiated cells were manually counted by identifying cells containing lipid droplets. The obtained data were normalized to adipocytes differentiated under 1g conditions. Additionally, images were analyzed for lipid droplet count and lipid size using the imfmdcircles function in MATLAB. These data were normalized to osteocytes differentiated at 1g. For both analyses, images from four positions per sample were acquired and quantified.

[0101] Quantitative Analysis of Osteogenic Differentiation. After osteogenic induction, cells were washed with PBS and stained with 2% Alizarin Red S solution and DAPI (1 : 10,000 dilution) for 5 minutes. The cells were then rinsed with PBS and imaged using a Lionheart FX automated microscope (BioTek, USA) with a 10x objective to visualize calcium deposits. The area of deposited calcium per image field was quantified using ImageJ (NIH) and normalized to the cell number, as indicated by DAPI staining. To quantify Alizarin Red S staining, 10% cetylpyridinium chloride (Sigma- Aldrich) was added, and cells were incubated for 20 minutes to elute the stain. The supernatants were then collected, and the absorbance of the eluted stain was measured at 570 nm using a plate reader (BioTek Hl). The obtained data were normalized to the cell number per well, as determined by DAPI staining.

[0102] Cryopreservation of Simulated Microgravity Exposed Adipocyte-Derived Stem Cells. Human adipose-derived stem cells (hADSCs) cultured under simulated microgravity (spG) on an RPM machine were harvested on Days 1 and 4 for cryopreservation. The cryopreservation medium consisted of 50% MesenPRO RS™ medium, 40% FBS, and 10% DMSO. A total of 5 x io5cells were resuspended in 1 mL of the prepared cry opreservation medium and transferred into a cryogenic vial. Controlled-rate freezing was performed by placing the cryogenic vials in an isopropanol freezing container, followed by overnight cooling at -80°C. The cells were then stored in a liquid nitrogen tank for one month before being tested for gene expression related to sternness and differentiation into adipocytes or osteocytes.

[0103] Statistical Analysis. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test for pairwise comparisons in Prism 10 (GraphPad Software Inc., San Diego, CA, USA. Statistical significance is denoted as follows: **ns = notsignificant, *p < 0.05, **p < 0.01, ***p < 0.001, **p < 0.0001. Experiments were performed using three different donors, each with four independent replicates.

[0104] Results and Discussion. Studying the effects of microgravity on stem cells is crucial for understanding their behavior in space and optimizing their potential for regenerative medicine. However, direct access to spaceflight experiments is limited, costly, and logistically challenging. To overcome these constraints, simulated microgravity models, such as the random positioning machine (RPM), provide an effective alternative by continuously altering the orientation of cell cultures, thereby eliminating the influence of directional gravity. While RPM-based models do not perfectly replicate true microgravity, they offer valuable insights into mechanotransduction, proliferation, and differentiation responses under reduced mechanical loading conditions.

[0105] Biphasic Proliferation and Stem Cell Marker Alterations in ADSCs Under Simulated Microgravity. To investigate the effects of simulated microgravity on adipose-derived stem cells (ADSCs), cells were expanded from 3 different donors, detached, and seeded into a microvessel system within a 4-well plate. The plates were then placed onto a random positioning machine (RPM), where ADSCs were cultured under simulated microgravity conditions for 1, 4, and 7 days. At each time point, cells were collected and analyzed for proliferation, morphology, and stem cell marker expression to assess their mechanosensitive responses. Additionally, cells were stored for subsequent differentiation assays, evaluating their potential for adipogenic and osteogenic commitment following microgravity exposure. Understanding how ADSCs respond to microgravity is essential for determining their suitability for regenerative applications in space and for identifying potential mechanisms regulating stem cell fate in mechanically unloaded environments. By assessing proliferation, morphological changes, and stem cell marker expression, this study aims to elucidate whether microgravity alters the expansion potential and sternness properties of ADSCs.

[0106] Phase-contrast images reveal significant morphological changes in ADSCs under simulated microgravity (s-pg). While cells cultured under normal gravity (1 g) exhibit their typical elongated, spindle-like morphology, those under s-pg appear more rounded with reduced cell spreading, indicating altered adhesion and cytoskeletal remodeling. These structural differences suggest that ADSCs experience challenges in maintaining adhesion under microgravity, which could influence their proliferation and survival. At Day 4, the ADSC population under s-pg appears denser than at Day 1, correlating with the proliferation peak observed in the quantification data. However, by Day 7, the confluency of ADSCs in s-pg is noticeably lower, suggesting that despite the initial increase in proliferation, long-term exposure to microgravity reduces cell adhesion and possibly leads to increased detachment. Given that stem cell attachment and expansion are critical for tissue regeneration, understanding the underlying mechanisms driving this biphasic proliferation response may provide insights into how mechanical cues influence ADSC behavior. Proliferation data further supports this observation, showing a biphasic growth response to microgravity. At Day 1, ADSC proliferation under s-pg is comparable to that in 1g conditions, indicating no immediate effect. By Day 4, a significant increase in cell proliferation is observed, suggesting that ADSCs initially respond to mechanical unloading with increased cell division, possibly due to altered cytoskeletal tension or metabolic shifts. This initial enhancement in proliferation might reflect a compensatory response to reduced mechanical constraints, enabling the cells to maximize growth potential in an environment where substrate interactions are limited. However, by Day 7, proliferation declines significantly, indicating that prolonged exposure to microgravity ultimately suppresses ADSC expansion, likely due to reduced adhesion, mechanotransduction deficiencies, or metabolic adaptation. The decrease in proliferation at later time points suggests that although ADSCs can initially adjust to the microgravity environment, extended exposure leads to limitations in their ability to sustain growth, potentially restricting their regenerative capacity in mechanically unloaded conditions.

[0107] To further examine how microgravity influences ADSC function, stem cell marker expression was analyzed to determine whether changes in proliferation and adhesion were associated with shifts in sternness characteristics. CD44, an adhesion and migration marker, follows a biphasic pattern, showing significant downregulation at Day 4 but recovering by Day 7. This suggests that ADSCs initially experience impaired adhesion under microgravity but later activate compensatory mechanisms to restore attachment and migration-related functions, reflecting a mechanoadaptive response. The ability of ADSCs to restore CD44 expression at later time points suggests that despite mechanical unloading, they retain some capacity to adjust their adhesion properties, which could be crucial for their longterm survival and function in microgravity conditions. In contrast, CD90, a key marker of multipotency and self-renewal, remains unchanged across all time points, indicating that ADSCs maintain their sternness under microgravity despite fluctuations in proliferation and adhesion-related changes. The stability of CD90 expression suggests that while microgravity influences growth dynamics, it does not compromise the fundamental identity of ADSCs, making them a potentially viable source for regenerative applications even in mechanicallyunloaded environments. However, the sustained downregulation of CD 105 at both Day 4 and Day 7 suggests a distinct response to microgravity that may have implications for lineage commitment. CD105, a TGF-P co-receptor involved in mesenchymal differentiation, is widely associated with endothelial differentiation potential in MSCs. Its persistent suppression under microgravity could indicate a shift away from a proliferative stem-like state and toward differentiation, possibly favoring adipogenic lineage commitment rather than impairing endothelial differentiation. While CD 105 is commonly linked to vascularization in MSCs, its role in ADSCs appears to be broader, involving proliferation, differentiation balance, and immunomodulation. Instead of being solely a vascularization marker in ADSCs, CD105 may act as a mechanosensitive regulator of ADSC fate, responding to mechanical unloading by altering TGF-P signaling pathways. The long-term suppression of CD 105 suggests that ADSCs in microgravity may have a reduced capacity to engage in endothelial differentiation, which could have implications for vascular regeneration in space environments. Future studies should explore whether the downregulation of CD 105 under microgravity correlates with increased adipogenic differentiation, changes in immunomodulatory potential, or shifts in other MSC differentiation pathways.

[0108] The hierarchical clustering heatmap (FIG. 23A) revealed segregation of gene expression patterns based on time point and culture condition; however, donor variability obscured clear distinctions. In contrast, tSNE (FIG. 23B) effectively resolved these differences, highlighting distinct sample clustering despite donor-to-donor variability. Unlike PCA, which captures global variance linearly, tSNE, a non-linear method, better preserves local relationships, making it more effective in distinguishing subtle biological differences. This is particularly relevant in transcriptome analysis, where donor variability introduces high-dimensional noise. By reducing the influence of dominant variation sources (e.g., donor effects) and emphasizing biologically relevant similarities, tSNE allows for better separation of experimental groups. To accurately assess the effects of simulated microgravity, we performed differentially expressed gene (DEG) analysis while controlling for donor variability. We applied batch effect correction using removeBatchEffect from the / / / 77 / 7 / apackage, adjusting for inter-donor differences while preserving true biological signals. This normalization ensured that transcriptional changes were primarily driven by experimental conditions rather than donor-specific variations. As shown in FIG. 23C, a Venn diagram illustrates the upregulated and downregulated genes, highlighting overlapping and unique transcriptional changes across comparisons. However, the number of differentiallyexpressed genes (DEGs) was minimal, suggesting that the transcriptional response to simulated microgravity remains relatively stable under these conditions.

[0109] We next conducted pathway analysis by categorizing transcriptional responses into three groups: early responses to simulated microgravity (Day 1 - Sug vs. Static), late responses (Day 4 - Sug vs. Static), and temporal changes over time (Day 4 - Sug vs. Day 1 - Sug). To investigate early responses, we analyzed DEGs between day 1 in simulated microgravity (Sug) and day 1 in static conditions (Static) (FIG. 23D). Gene Ontology (GO) enrichment analysis revealed significant downregulation of genes involved in cytoskeletal organization, synapse-associated structures, and insulin-like growth factor binding. The enriched pathways included actin filament organization and postsynaptic cytoskeleton organization, suggesting that ADSCs rapidly respond to mechanical cues by reducing cytoskeletal activity in the early stages. Conversely, genes associated with acyl-CoA desaturase activity were upregulated, indicating an early activation of lipid metabolism in response to mechanical stimuli, potentially as a compensatory mechanism to meet energy demands in simulated microgravity.

[0110] To assess temporal changes in simulated microgravity, we compared gene expression between day 4 and day 1 under microgravity conditions (Day 4 - Sug vs. Day 1 - Sug) (FIG. 23E). This analysis revealed a pronounced metabolic shift, with upregulation of steroid, lipid, sterol, and cholesterol biosynthesis pathways, as well as increased oxidoreductase activity. These changes suggest that ADSCs adapt to prolonged mechanical unloading by prioritizing lipid metabolism and steroid biosynthesis to maintain cellular homeostasis and membrane integrity. In contrast, genes associated with cell mobility, migration, collagen binding, heparan binding, actin binding, integrin binding, cell adhesion molecule binding, and extracellular matrix structural components were significantly downregulated, indicating a progressive decline in adhesion, extracellular interactions, and cytoskeletal organization. As microgravity exposure continued, ADSCs exhibited a late-stage adaptation characterized by metabolic reprogramming and diminished structural integrity. The upregulation of sterol -related pathways, particularly C-5 sterol desaturase activity, suggests a role in cholesterol biosynthesis and membrane remodeling — key processes for maintaining plasma membrane stability, lipid raft formation, and intracellular signaling, all of which are critical for stem cell self-renewal and fate determination. The concurrent increase in oxidoreductase activity suggests enhanced redox balance and metabolic flexibility, supporting ADSC survival under low-gravity conditions. These findings indicate that whilemicrogravity promotes metabolic resilience in ADSCs, it simultaneously weakens their adhesive and migratory properties, potentially altering their functional capacity over time. [OHl] A direct comparison of gene expression between day 4 and day 1 under simulated microgravity conditions (FIG. 23F) further highlighted significant transcriptional changes, particularly in metabolism, adhesion, and extracellular interactions. Cellular components associated with extracellular exosomes, focal adhesion, and respiratory chain complexes were upregulated, suggesting increased intercellular communication and enhanced mitochondrial activity in response to prolonged mechanical unloading. Additionally, genes involved in oxidative phosphorylation, cholesterol biosynthesis, ATP metabolic processes, sterol biosynthesis, and response to type I interferon were upregulated, indicating a metabolic shift prioritizing energy production, lipid metabolism, and immune regulation. These adaptations likely help maintain cellular homeostasis in microgravity. In contrast, pathways regulating cell adhesion, integrin-mediated signaling, and cellular response to copper ions were downregulated, reflecting a progressive decline in adhesion properties and extracellular matrix interactions. This suggests that while ADSCs enhance metabolic resilience through increased mitochondrial function and lipid biosynthesis, they simultaneously exhibit diminished adhesion and mechanotransduction capabilities. These findings further support the idea that microgravity reprograms ADSC function, promoting metabolic adaptation at the expense of structural integrity and adhesion-dependent signaling.

[0112] Conclusion. These findings suggest that microgravity induces a biphasic response in ADSCs, where short-term proliferation is enhanced (Day 4), but long-term growth is inhibited (Day 7), possibly due to reduced adhesion and impaired mechanotransduction. The transient downregulation and recovery of CD44 indicate that ADSCs undergo mechanoadaptation, where initial adhesion-related challenges are eventually counteracted by cellular compensatory mechanisms. However, the sustained downregulation of CD 105 suggests that microgravity imposes long-term limitations on endothelial differentiation, which could have significant implications for vascular tissue engineering in space. The stable expression of CD90 further confirms that the fundamental sternness of ADSCs is preserved, despite mechanical and proliferative shifts. Given these findings, further research is needed to determine how mechanical unloading influences ADSC differentiation potential and whether specific interventions, such as biochemical signaling modulation or scaffold-based mechanical support, could enhance their regenerative potential under microgravity conditions. Understanding how ADSCs dynamically respond to mechanicalunloading is crucial for optimizing their applications in space medicine and regenerative therapies on Earth.EXAMPLE 3

[0113] This example provides a description of methods of the present disclosure.

[0114] Visual representations demonstrate a progressive increase in the number of cells over time, quantified as a percentage change in cell count. Proliferating cells are quantified through the staining of ki-67-positive cells, indicating that exposure to pG for tl and t2 results in an enhancement of proliferation, thereby explaining the observed increase in cell count. Dead cells remain consistent, suggesting the absence of cell death when comparing microgravity-exposed and non-microgravity-exposed cells. DNA damage is measurable when cells are exposed to microgravity at early exposure durations, but not for longer exposures. This could indicate a stabilization of damaged cells or the possibility that some damaged cells have died and not been analyzed. See FIG. 20.

[0115] Enhanced microgravity exposed stem cells are triggered to differentiate into fats using commercial kits. By measuring Nile Red positive cells, an increase in number of fat cells derived from adiposed derived stem cells can be seen, measured to be 2X minimal increament. Quantifying the lipid droplets using imaged-based approaches shows no changes in number of lipids / cell or the size of the lipids.

[0116] Enhanced microgravity exposure of stem cells induces differentiation into bone cells through commercial kits. By analyzing alizarin (calcium deposition)-positive cells, which serve as late-stage markers of osteogenic differentiation, we observe a significant increase in the number of bone-like cells derived from adipose-derived stem cells. This differentiation is accompanied by a twofold minimal increment in mineralization activity. Similarly, quantifying alkaline phosphatase, an early-stage marker of osteogenic differentiation, reveals a substantial increase in the number of cells expressing ALP and a corresponding rise in ALP expression, approximately 2.0 times higher compared to cells not exposed to microgravity.

[0117] To date, results demonstrating increased proliferation, enhanced genetic pluripotency markers, and verified differentiation activity have been presented. It was further sought to ascertain whether these cells can be maintained in an enhanced state. These findings indicate that they do retain improved potency after a month of cry opreservation.

[0118] RNA-sequencing followed by transcriptomics analyses was performed to understand the mechanisms of the observations herein.> 90 >

[0119] tSNE shows distinguishing subtle biological differences and Venn diagram illustrates the upregulated and downregulated genes, highlighting overlapping and unique transcriptional changes across comparisons. However, the number of differentially expressed genes (DEGs) was minimal, suggesting that the transcriptional response to simulated microgravity remains relatively stable under these conditions.

[0120] Early response to simulated microgravity (tl), from a mechanobiology perspective, shows reduced developmental (lineage) progression, exhibits disrupted structural organization. Late response to simulate dmicrogravity (t2), from a mechanobiology perspective, lose traditional adhesion and structural cues, enter a primed or transitional state that might delay differentiation but enhance responsiveness to external cues. Temporal changes when exposed to simulate dmicrogravity (t2 vs tl), from a mechanobiology perspective, lose structural cues (adhesion, integrin signaling), possibly shifting toward a more undifferentiated or migratory phenotype.

[0121] Mechanobiology and transcriptomic analyses revealed cytoskeletal, adhesion, migratory changes, lamin was examined to provide protein-level mechanistic insights. Lamin is an intermediate filament protein that plays a central role in mechanotransduction and in stem cells lamin is crucial for maintaining identity, differentiation capacity, and nuclear integrity. From ADSC exposed and not exposed to microgravity, it was observed that in nonmicrogravity exposed ADSC, there exist healthy lamin enveloping the nucleus, and this diminishes when there is exposure to spG.

[0122] Lamin and the morphological roundness of nucleus is closely connected, where the protein plays a structural and mechanosensory role that directly influences nuclear shape and stiffness.

[0123] Furthermore, in stem cells, roundness is an indicator of cell health, differentiation state, and mechanotransduction sensitivity. It was observed that high lamin levels around the nucleus shows high nuclear aspect ratio, and the opposite effect is expected, a rounder nucleus (aspect ratio close to 1) is a result of less lamin around the nucleus, which could explain the higher sternness and differentiation potential we measured. See FIG. 21.

[0124] Reduced Lamin can also increase DNA damage susceptibility, which was measured to be true from the studies herein.

[0125] FIG. 21 (formerly slide 17) presents data indicating that exposure of cells to microgravity induces distinct morphological and nuclear changes. Specifically, a reduction in Lamin A / C expression was observed at the nuclear periphery, as confirmed by immunofluorescence imaging and quantification of the nuclear / perinuclear Lamin A / Cintensity ratio. Notably, this reduction in Lamin A / C was accompanied by an upregulation of sternness-associated markers such as Nanog and Oct4, suggesting that mechanical unloading under microgravity promotes a more stem-like state. This observation aligns with previous reports showing that reduced Lamin A / C levels can enhance stem cell differentiation potential. Although some increase in DNA damage markers (H2AX) were detected, the extent was minimal — less than 5% — and therefore not a major concern in this context.

[0126] Stem cells exposed to simulated microgravity undergo the following: a metabolic reprogramming to support survival; loss of structural cues (adhesion, integrin signaling), possibly shifting toward a more undifferentiated or migratory phenotype; upregulation of secretion and vesicle pathways, suggesting a high-communication state, potentially to modulate their environment or compensate for impaired contact-based signaling. Non-mechanobiological studies make microgravity a valuable model for studying plasticity, metabolic resilience, and niche-independent survival in stem cells.

[0127] Although the present disclosure has been described with respect to one or more particular embodiments and / or examples, it will be understood that other embodiments and / or examples of the present disclosure may be made without departing from the scope of the present disclosure._ 7 1

Claims

CLAIMS1. A method for improving stem cell differentiation potency in a stem cell population comprising culturing the stem cell population under microgravity for about 12 hours to about 36 hours, wherein at least 70% of the stem cell population can further differentiate under differentiation conditions.

2. The method according to claim 1, wherein the stem cell population is cultured in a welled plate comprising a microvessel.

3. The method according to claim 2, wherein the microvessel is made from polydimethylsiloxane.

4. The method according to claim 1, wherein the microgravity occurs from culturing the stem cell population on a random position machine.

5. The method according to claim 4, wherein random position machine moves at an average velocity of 45 to 75 deg / s.

6. The method according to claim 4, wherein the random position machine moves in one or more random directions.

7. The method according to claim 1, wherein the stem cells are human adipose-derived stem cells, bone marrow-derived stem cells, or induced pluripotent stem cells.

8. The method according to claim 1, wherein at least 80% of the stem cell population can further differentiate under differentiation conditions.

9. The method according to claim 8, wherein at least 85% of the stem cell population can further differentiate under differentiation conditions.

10. The method according to claim 9, wherein at least 90% of the stem cell population can further differentiate under differentiation conditions._ 9 _11. The method according to claim 1, wherein the stem cell population was cultured for about 24 hours to allow for cell adherence prior to culturing under microgravity.

12. The method according to claim 1, wherein the stem cells are cultured under microgravity for about 15 hours to about 30 hours.

13. The method according to claim 12, wherein the stem cells are cultured under microgravity for about 20 hours to about 28 hours.

14. The method according to claim 13, wherein the stem cells are cultured under microgravity for about 24 hours.

15. The method for differentiating stem cells with an improved differentiation potency, comprising contacting the stem cells improved by a method of claim 1 with a differentiation medium for a period of time such that stem cells are differentiated to the desired cell type.

16. The method according to claim 15, wherein the differentiation medium is exchanged at least every three days.

17. The method according to claim 15, wherein the stem cells are differentiated for at least 14 days.

18. The method according to claim 17, wherein the stem cells are differentiated for at least 21 days.> 77 >

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