Generation of tissue-specific stem cells from somatic cells

Abstract

A method of generating adult stem cells from somatic cells is provided. The method involves isolating somatic cells from a donor tissue and culturing the isolated cells in a tissue-specific growth medium. The cultured cells are subjected to proteolytic detachments once they reach a sufficient level of confluency. The confluent somatic cells are treated a culture of somatic cells isolated from a donor tissue with a combination of growth factors and / or chemical compounds. The cells are then reprogrammed by subjecting the cells to a suspension culture treatment without any substrate or beads to attach and are forced to rely on cell-to-cell contact. This treatment leads to formation of aggregates comprising cells that display stem-cell like characteristics.

Description

1 GENERATION OF TISSUE-SPECIFIC STEM CELLS FROM SOMATIC CELLS FIELD OF THE INVENTION

[0001] The present invention relates to biotechnology and cellular biology, particularly methods for generating stem cells, specifically non-embryonic stem cells, also known as somatic stem cells or adult stem cells (ASCs). Specifically, the invention describes a method of generating tissuespecific adult stem cells from somatic cells without the use of transgenes or viral vectors, thus offering a safer and more efficient approach for therapeutic applications, regenerative medicine, disease modeling, and tissue engineering, circumventing the risks associated with genetic modifications and viral integration. Kits for the proposed technique and cells generated by the method are also disclosed. BACKGROUND OF THE INVENTION

[0002] Stem cell therapy has emerged as a promising frontier in regenerative medicine, offering the potential to replace damaged or diseased tissues with healthy, functioning cells. Among the various types of stem cells, induced pluripotent stem cells (iPSCs) have garnered considerable attention due to their remarkable ability to differentiate into virtually any cell type in the human body. However, despite their promise, significant safety concerns hinder their translation into mainstream clinical therapies. These concerns primarily stem from the potential for tumorigenesis caused by residual undifferentiated cells and the genetic and epigenetic instability introduced by transgenes such as OCT4, SOX2, KLF4, and c-MYC, which are typically delivered via viral vectors. Such complications present formidable barriers to the widespread clinical application of iPSCs.

[0003] Adult stem cells (ASCs), also known as tissue-specific stem cells, represent a distinct and rare population of undifferentiated cells residing within adult tissues in various organs. These cells possess two key properties: the ability to self-renew and multipotency, meaning they can differentiate into a limited number of specialized cell types that contribute to the maintenance and repair of the tissue in which they are found. Unlike iPSCs, ASCs naturally participate in the regeneration of tissues and organs without the associated risks of tumor formation or genetic manipulation, making them an attractive alternative for therapeutic applications. However, their rarity in tissues poses a significant challenge, as the isolation and expansion of sufficient numbers for clinical use remain difficult.2 BRIEF DESCRIPTION OF FIGURES

[0004] The above and other aspects, features and advantages of the invention will become more readily apparent from the following description, reference being made to the accompanying drawings in which:

[0005] Figure 1 shows that mouse TEFs grown in suspension culture display MSC surface markers while undergoing detachment-induced apoptosis (anoikis).

[0006] Figure 2 shows that mouse TEF-derived spheroids grown in SIST express mMSC markers, proliferate and exhibit self-renewal capacity.

[0007] Figure 3 shows that mouse TEF-derived spheroids are multipotent with no tumorigenic potential.

[0008] Figure 4 shows characterization of mouse TEF-derived spheroids vs. monolayer via differential expression gene analysis.

[0009] Figure 5 shows that human dermal fibroblast- and hepatocyte-derived spheroids possess SC properties.

[0010] Figure 6 shows differential capacity of human hepatocyte-derived spheroids and formation of spheroids by human keratinocytes that proliferate and express keratinocyte-specific stem cell markers.

[0011] Figure 7 shows mouse TEFs grown in suspension culture and expressing stem cell markers undergo cell death.

[0012] Figure 8 shows that mouse TEF-derived spheroids are highly proliferative.

[0013] Figure 9 shows transcriptome analysis using RNA-seq in TEFs cultured as a monolayer and spheroids.

[0014] Figure 10 shows expression distribution for selected mouse MSCs-specific genes.

[0015] Figure 11 shows expression distribution of selected ECM genes in monolayer and spheroid cells.3

[0016] Figure 12 shows expression distribution for self-renewal genes in monolayer and spheroid cells.

[0017] Figure 13 shows feature plots of expression distribution for Hox and Mmp genes.

[0018] Figure 14 show Hox and Mmp genes are highly expressed in cluster 7.

[0019] Figure 15 show that human dermal fibroblast-derived spheroids are proliferative.

[0020] Figure 16 illustrates the procedure for generating human cartilage-producing cells (chondrocytes) from human skin fibroblasts.

[0021] Figure 17 shows Alcian blue staining of chondrocytes generated from human skin fibroblasts using the suspension culture method, with the chondrocytes staining positive for Alcian blue, in contrast to the human skin fibroblast control, which stained negative.

[0022] Figure 18 illustrates the characterization of spheroid-derived chondrocytes generated using suspension culture method, through immunofluorescence staining, using specific markers for human chondrocytes. SUMMARY OF THE INVENTION

[0023] There is an urgent need for a safer, non-transgenic, and virus-free approach to generate large quantities of ASCs for therapeutic purposes. The present invention addresses this need by providing a transgene-free and virus-free method for generating tissue-specific adult stem cells from somatic cells, offering a reliable source of ASCs for stem cell-based therapies.

[0024] The present invention provides a method for the direct generation of high-yield, tissuespecific adult stem cells from somatic cells without the use of transgenes or viral vectors. The method described in this invention is based on the discovery that primary cells, irrespective of their lineage (mesoderm, endoderm, or ectoderm), undergo a distinct morphological transformation and exhibit the expression of stem cell-associated markers of their origin tissue when they are detached from the cell culture plate and remain suspended in the culture medium. This process occurs simultaneously with detachment-induced apoptosis, also known as anoikis. This method utilizes specific culture conditions and chemical factors to reprogram somatic cells directly into adult stem cells of the desired tissue type.4

[0025] This method offers several key advantages over existing technologies: ™ No genetic modification: The proposed method does not require the introduction of exogenous transgenes, thus reducing the risk of genetic instability or tumorigenesis. ™ No viral integration: By avoiding the use of viral vectors, this approach eliminates concerns about viral integration into the host genome. ™ Scalability: The process can be scaled for clinical-grade production of stem cells for therapeutic use. ™ Tissue specificity: The method allows for the generation of adult stem cells tailored to specific tissue types, increasing their relevance for targeted regenerative therapies.

[0026] The invention is applicable to a wide range of somatic cells across three germ layers, mesoderm, ectoderm and endoderm, including but not limited to fibroblasts, keratinocytes, and hepatocytes. As a result, it offers a safer, more effective technique for generating patient-specific adult stem cells for use in regenerative medicine, disease modeling, and personalized medicine. DETAILED DESCRIPTION

[0027] The following description is of preferred embodiments by way of example only and without limitation to the combination of features necessary for carrying the invention into effect.

[0028] All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0029] Although various features of the present disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment.

[0030] The following definitions supplement those in the art and are directed to the current application. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.5

[0031] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0032] In this application, the use of "or" means "and / or" unless stated otherwise. The terms "and / or" and "any combination thereof and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any and all combinations are specifically contemplated. The term "or" can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

[0033] Furthermore, use of the term "including" as well as other forms, such as "include", "includes," and "included," is not limiting.

[0034] Reference in the specification to "some embodiments," "an embodiment," "one embodiment" “alternate embodiment”, or "other embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.

[0035] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or openended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0036] The term "about" in relation to a reference numerical value and its grammatical equivalents as used herein can include the numerical value itself and a range of values plus or minus 10% from that numerical value. The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, per the6 practice in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In another example, the amount "about 10" includes 10 and any amounts from 9 to 11. Embodiments

[0037] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0038] In an embodiment of the invention, the invention describes a method of generating adult stem cells from somatic cells. The method comprises the steps of treating a culture of somatic cells isolated from a donor tissue. The isolated somatic cells are treated with a combination of growth factors and / or chemical compounds. The method further comprises inducing reprogramming of the isolated somatic cells by promoting cell to cell contact. Cell to cell contact allows the isolated somatic cells to generate or form aggregates comprising adult stem cells.

[0039] In a further embodiment, the invention describes that reprogramming of somatic cells occurs by inducing a stem-cell like state in the isolated somatic cells through cell-to-cell contact.

[0040] In a further embodiment, the invention describes that reprogramming of somatic cells is induced by subjecting the somatic cells to a suspension culture technique. The suspension culture technique allows optimal cell-cell contact by keeping the isolated somatic cells afloat. Specifically, the culture technique is devoid of any membrane or substate to attach on thereby promoting cell7 cell attachment to form aggregates / spheroids. The suspension culture technique increases / promotes cell-to-cell contact.

[0041] In a further embodiment, the suspension culture technique employed is a hanging drop culture technique, g-force culture technique, centrifugation culture technique, shaker flask culture technique, spinner flask culture technique, agitation culture technique, closed continuous culture technique, open continuous culture technique, gravity-controlled culture technique, or magnetic stirrer culture technique.

[0042] In a further embodiment, the invention describes that aggregates comprising adult stem cells display stem cell like characteristics.

[0043] In a further embodiment, the invention describes that the isolated somatic cells form aggregates of adult stem cells from which they were originally derived. For instance, if the isolated somatic cells (e.g. fibroblasts) were derived from mesenchymal stem cells, the somatic cells when subjected to cell-cell contact forms aggregates of cells that display mesenchymal stem cell like properties, similarly if the isolated somatic cells (e.g. hepatocytes) were originally derived from hepatic stem cells, the somatic cells when subjected to cell-cell contact forms aggregates of cells that display hepatic stem cell like properties

[0044] In a further embodiment, the invention describes that the isolated somatic cells are from mesoderm / mesodermal donor tissue the cells form aggregates of mesodermal stem cells, if the isolated somatic cells are from ectoderm / ectodermal donor tissue the cells form aggregates of ectodermal stem cells, and if the isolated somatic cells are from the endoderm / endodermal donor tissue the cells form aggregates of endodermal stem cells.

[0045] In a further embodiment, the invention describes that the aggregates of adult stem cells display stem cell like characteristics such as self-renewal capacity and expression of tissue specific surface markers.

[0046] In a further embodiment, the invention describes that expression of surface markers is determined by immunofluorescence staining.8

[0047] In a further embodiment, the invention describes that mesodermal stem cells express mesodermal tissue specific surface markers such as CD73, CD105, CD106, CD146, CD166, or STRO-1. The endodermal stem cells express ectodermal tissue-specific surface markers such as CD117, CD133, or EPCAM. The ectodermal stem cells express tissue-specific surface markers such as TfR / CD71 or p63 / TP73L.

[0048] In a further embodiment, the proposed method further comprises a step of subjecting the aggregates of adult stem cells to predefined growth conditions to promote differentiation into a target-tissue. The predefined growth conditions may be target-tissue specific. For instance, the predefined growth conditions for endodermal somatic cells may be set to promote reprogramming of the endodermal cells to form aggregates of endodermal stem cells.

[0049] In a further embodiment, the proposed method may further comprise target-tissue specific growth conditions such as a target-tissue specific differentiation medium with essential nutrients and growth factors that induces the stem cells to differentiate into a target tissue.

[0050] In a further embodiment, the differentiation of stem cells to a target-tissue is confirmed by the expression of target tissue-specific proteins, e.g. mesodermal cellular proteins, endodermal cellular proteins etc.

[0051] In a further embodiment, the invention describes that aggregates of mesodermal stem cells differentiate to form a mesodermal tissue, aggregates of ectodermal stem cells differentiate to form an ectodermal tissue, and aggregates of endodermal stem cells differentiate to form an endodermal tissue.

[0052] In a further embodiment, the invention suggests that the isolated stem cells form aggregate of various different shapes, e.g. spheroids.

[0053] In a further embodiment, the proposed method further comprises the steps of isolating somatic cells from the donor tissue, followed by culturing the isolated somatic cells in a suitable growth medium, allowing the isolated somatic cells to grow in the growth medium until they reach optimal confluency; and detaching the confluent somatic cells by treating with a proteolytic enzyme. The proteolytic enzyme may be bromelain, chymotrypsin, ficin, papain, serrapeptase,9 and trypsin. These steps are generally performed prior to treating the isolated somatic cells with the combination of growth factors and / or chemical compounds.

[0054] In a further embodiment, the invention proposes carrying out the steps of treating the isolated stem cells with growth factors / chemical compounds and inducing reprogramming of the cells immediately, or within a few minutes, or within an hour from proteolytic detachment of the confluent cells.

[0055] In a further embodiment, the invention describes that the isolated somatic cells may be mesodermal somatic cells, osteoblasts, adipocytes, chondrocytes, myocytes, fibroblasts, endodermal somatic cells, hepatic stellate cells, skeletal muscle cells, satellite cells, beta cells, ectodermal somatic cells, keratinocytes, or a combination thereof.

[0056] In a further embodiment, the invention describes that the donor tissue is an ectodermal tissue (such as skin tissue), a mesodermal tissue (such as adipose tissue, muscle tissue), or an endodermal tissue (such as liver tissue, a pancreatic tissue), or a combination thereof.

[0057] In a further embodiment, the invention describes that the growth factors may be human epidermal growth factor (hEGF), basic fibroblast growth factor (bFGF), human insulin-like growth factor-1 (hIGF-1), rho-associated coiled-coil inhibitor (ROCKi), human hepatocyte growth factor (hHGF), mesodermal-specific growth factors, endodermal-specific growth factors, ectodermalspecific growth factors or a mixture of any of the above.

[0058] In a further embodiment, the invention describes that the chemical compound is rhoassociated coiled-coil-containing protein kinase (ROCK) inhibitor, mesodermal-specific chemical compound, endodermal-specific chemical compound, ectodermal-specific chemical compound or a mixture of any of the above.

[0059] In a further embodiment, the invention describes that the isolated somatic cells are maintained in the growth medium at optimal culture conditions.

[0060] In a further embodiment, the invention describes that the optimal culture conditions comprise a pH ranging from 5.5-7.5, a temperature ranging from 35’C - 40’C, and a growth medium comprising 3-7% CO2, 19-23% O2, and 72-76% N210

[0061] In a further embodiment, the invention describes that the growth medium may be DMEM / F-12 supplemented with 10% Fetal bovine serum (FBS), Williams' Medium E supplemented with 10% Fetal bovine serum (FBS), Williams' Medium E supplemented with 10% Fetal bovine serum (FBS) and additive nutrients, keratinocyte serum free medium (k-sfm), or 8UO>OLK` ?KJOZR XZUUQKRKSYKJ \OYN / * e? IGQIOZR LWTR :<56A GSJ +*# 9KYGQ HT[OSK XKWZR (FBS), a culture medium supplemented with MesenCult™-ACF Chondrogenic Differentiation Kit, any culture medium comprising essential nutrients for the growth of somatic cells, or a serum-free medium comprising essential nutrients for the growth of somatic cells.

[0062] In a further embodiment, the invention describes that the optimal confluency ranges from 60%-95%, preferably between 70% - 90%.

[0063] In a further embodiment, the invention describes that the differentiation medium may be DMEM / F-12 supplemented with 10% Fetal bovine serum (FBS), Williams' Medium E supplemented with 10% Fetal bovine serum (FBS), Williams' Medium E supplemented with 10% Fetal bovine serum (FBS) and additive nutrients, keratinocyte serum free medium (k-sfm), or 8UO>OLK` ?KJOZR XZUUQKRKSYKJ \OYN / * e? IGQIOZR LWTR :<56A GSJ +*# 9KYGQ HT[OSK XKWZR (FBS), a culture medium supplemented with MesenCult™-ACF Chondrogenic Differentiation Kit, any culture medium that supports differentiation of endodermal, ectodermal or mesodermal cells, or a serum-free medium mesodermal-specific growth factors, endodermal-specific growth factors, ectodermal-specific growth factors or a mixture of any of the above.

[0064] In a further embodiment, the invention describes that the isolated somatic cells are treated with a combination of target tissue-specific growth factors, e.g. endodermal tissue specific growth factors, or mesodermal tissue specific growth factors.

[0065] In a further embodiment, the invention describes that the method is for generating an ectodermal tissue (such as skin tissue, epithelial lining of mouth, tooth enamel etc.) from isolated ectodermal somatic cells, an endodermal tissue (such as epithelial lining of respiratory system, liver tissue, pancreatic tissue etc.) from isolated endodermal somatic cells or a mesodermal tissue (such as muscle tissue, notochord skeletal tissue, lymphatic system tissue etc.) from isolated mesodermal somatic cells.11

[0066] In a further embodiment, the invention describes that the proposed method can be used for tissue-replacement treatment, for tissue repair treatment, for stem-cell research, for genetic research, for drug testing, for disease modelling, for stem cell therapy, or for developing a stem cell bank.

[0067] In an alternate embodiment, adult stem cells generated from the isolated somatic cells using the method described hereinbefore are provided.

[0068] In a further embodiment, use of the adult stem cells is disclosed for tissue-replacement treatment, for tissue repair treatment, for stem-cell research, for genetic research, for drug testing, for disease modelling, for stem cell therapy, or for developing a stem cell bank.

[0069] In a further embodiment, use of isolated somatic cells from a donor tissue for generating an ectodermal tissue (such as skin tissue, epithelial lining of mouth, tooth enamel etc.) from isolated ectodermal somatic cells, an endodermal tissue (such as epithelial lining of respiratory system, liver tissue, pancreatic tissue etc.) from isolated endodermal somatic cells or a mesodermal tissue (such as muscle tissue, notochord skeletal tissue, lymphatic system tissue etc.) from isolated mesodermal somatic cells using the method described hereinbefore is also provided.

[0070] In a further embodiment, a kit for generating adult stem cells or a target tissue is provided. The kit comprises a culture of confluent isolated somatic cells, a growth medium for supporting the growth of isolated somatic cells; an apparatus for suspension culture technique such as hanging drop or g-force centrifugation; and a differentiation medium for promoting differentiation of the isolated somatic cells to the adult stem cells or the target tissue.

[0071] In a further embodiment, a method of generating adult stem cells for treating stem cell disorders, for treating tissue damage, for treating genetic disorders, or for treating diseases, using the method of generating adult stem cells described hereinbefore is provided. Proposed technique

[0072] The method described herein allows for the generation of adult stem cells directly from somatic cells using a transgene-free, virus-free approach. The method involves the following steps:12

[0073] Somatic Cell Isolation: Human somatic cells, such as fibroblasts, keratinocytes, or hepatocytes, are isolated from donor tissue using standard tissue biopsy techniques, with minimal tissue damage and preservation of cell viability. Somatic cells can be isolated from a variety of tissues in the human body such as skin tissue for fibroblasts and keratinocytes. adipose tissue for adipocytes and mesenchymal stem cells, liver tissue for hepatocytes and hepatic stellate cells, muscle for skeletal muscle cells and satellite cells, pancreatic tissue for beta cells, and various other tissues. All the cells in the body are somatic, with the exception of gametes, which are reproductive cells, and therefore, somatic cells can be isolated from various other tissues in the body.

[0074] For fibroblasts, skin tissue is commonly used as the source, where a small skin biopsy is processed to release individual cells. Similarly, skin tissue is also used as a common source for Keratinocytes and liver tissue for hepatocytes. Once isolated, these cells are cultured under sterile conditions in a suitable growth medium or culture medium that provides the necessary nutrients, growth factors, and environmental conditions to promote cell proliferation. Some non-limiting examples of growth mediums: a) Fibroblasts can be cultured in DMEM / F-12, supplemented with 10% Fetal bovine serum (FBS), b) Hepatocytes can be cultures, Williams' Medium E, supplemented with 10% Fetal bovine serum (FBS), supplemented with additive nutrient for example, HepExtend™ Supplement or Hepatocyte Maintenance Supplement Pack from ThermoFisher, c) Keratinocytes can be cultured in keratinocyte serum free medium (k-sfm), or 8UO>OLK` ?KJOZR& \OYN / * e? IGQIOZR LWTR :<56A& XZUUQKRKSYKJ \OYN +*# 9KYGQ HT[OSK XKWZR (FBS). Many other culture mediums known in the art can be used for this purpose. Several serumfree mediums can also be used for culturing somatic cells. The various nutrients / growth factors and conditions required for cell proliferation, at least for fibroblasts, keratinocytes, or hepatocytes, and other somatic cels have been studied well. Therefore, any growth medium known for culturing somatic cells can be used for the proposed technique. The culture conditions are carefully monitored to maintain optimal pH, temperature, and oxygen levels, ensuring the cells remain healthy and viable. Generally, the cells are maintained in 5% CO2, 21% O2, and 74% N2 at 37’C. However, the optimal culture conditions can be varied as needed. The cells are allowed to grow until they reach a sufficient level of confluency, (typically between 70-90%), at which point they are ready for the next step in the protocol.

[0075] Induction of Reprogramming Through Suspension Culture Methods Promoting Cell–Cell Contact: The somatic cells are treated for 2–6 hours with a specific combination of growth factors,13 tailored to the target tissue type. For instance, fibroblasts may require a mixture of human epidermal growth factor (hEGF), basic fibroblast growth factor (bFGF), and human insulin-like growth factor-1 (hIGF-1), to promote cell proliferation. For instance, human Keratinocytes may require a mixture of human epidermal growth factor (hEGF), human basic fibroblast growth factor (bFGF) and human insulin-like growth factor 1 (hIGF-1), plus rho-associated, coiled-coil inhibitor (ROCKi). For instance, human Hepatocytes may require a mixture of human hepatocyte growth factor (hHGF), human basic fibroblast growth factor (bFGF) and human insulin-like growth factor 1 (hIGF-1), plus rho-associated, coiled-coil inhibitor (ROCKi). Along with these growth factors, chemical compounds such as Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor are also introduced to prevent apoptosis, ensuring the survival and resilience of the somatic cells during the reprogramming process.

[0076] Following this treatment, the cells are transferred to a novel suspension culture system specifically designed to facilitate physical interaction between the cells. This system is distinctly different from differs significantly from existing systems that use microcarriers, such as microbeads, which provide an anchorage or attachment surface for suspended cell cultures. In the proposed system, there is no attachment surface involved; instead, cells are brought into physical contact solely through suspension. The contact between the cells is achieved using one of two methods, both yielding comparable results, which promote direct cellular interactions without the need for external substrates. (A) Hanging Drop Suspension Culture: In this method, gravity encourages cell–cell contact, facilitating spheroid formation. Following the treatment the cells are seeded at a density of 75-200 cells per 20-40 µL drop of culture medium on the inner side of a culture petri dish, for instance 100 cells per 25 µL drop of culture medium on the inner side of a 100 mm tissue culture petri dish lid. The culture medium contained a specific combination of growth factors as mentioned above, tailored to the target tissue type, as described earlier. As mentioned above, for instance, for human fibroblasts and keratinocytes a mixture of human epidermal growth factor (hEGF), human basic fibroblast growth factor (bFGF) and human insulin-like growth factor 1 (hIGF-1), plus rhoassociated, coiled-coil inhibitor (ROCKi) can be used. For human keratinocytes, human hepatocyte growth factor (hHGF), human basic fibroblast growth factor (bFGF) and human insulin-like growth factor 1 (hIGF-1), plus rho-associated, coiled-coil inhibitor (ROCKi) can be used. The lid was carefully inverted (creating hanging drop) and placed atop a Petri dish filled with 5 mL of sterile Phosphate-buffered saline (PBS) or any other isotonic buffer. The cells were14 cultured in this inverted position within individual drops for 2 days. On day 3, the aggregated cells were transferred to ultra-low-attachment (ULA) 96-well flat-bottom plate, with each well containing 200 µL of the same culture medium. The cells were maintained in this system within a controlled incubator for 7 days, with half of the culture medium being refreshed every other day to ensure a consistent supply of nutrients and growth factors, thereby maintaining optimal culture conditions. (B) Spheroid Formation Using G-Force via Centrifugation: In this method, G-force generated by centrifugation promotes cell–cell contact, facilitating spheroid formation. After treatment, the cells are transferred to a suspension culture system, such as ULA 96-well round-bottom plates, or any other round bottom ultra-low attachment plates such as Corning® Elplasia® with 6-, 24- and 96- well formats from Corning or AggreWell™400 or AggreWell™800. The cells are transferred at a density of 500-1500 cells per well in 200-300 µL of culture medium, for instance 500 cells per well in 200 µL of culture medium. The plates are centrifuged at 300-200× g for 3-6 minutes, for instance 250× g for 3 minutes to precipitate the cells at the bottom of each well. This setup provides a nonadherent environment, mimicking the in vivo microenvironment by preventing attachment to the culture surface and encouraging cell–cell interactions, aided by the round-bottom design of the plates, which promotes spheroid formation. The cells are cultured in this system within a controlled incubator for 7 days, with half of the culture medium being refreshed every other day. Both suspension culture systems described above are crucial for inducing a stem-like state in somatic cells, promoting growth and facilitating the generation of cells in spheroid form. These spheroids display key characteristics of adult stem cells, including self-renewal capacity and the expression of specific surface markers, for instance the specific surface markers for human fibroblast–derived mesenchymal spheroids, or human keratinocytes–derived spheroids or human hepatocyte–derived spheroids as shown in the figures. Spheroid formation in this context results in a high yield of tissue-specific adult stem cells, which are primed for differentiation into functional cell types relevant to the target tissue.

[0077] The above-noted techniques are merely exemplary. Any technique known in the art that increases or promotes cell-cell contact thereby allowing spheroid formation can be employed here.

[0078] Stem Cell Confirmation: Following sphere formation and growth in suspension culture for 7 days, the cells within the spheroids are subjected to comprehensive analysis to confirm their15 identity as tissue-specific adult stem cells. This analysis involves assessing the expression of key stem cell surface markers that are characteristic of the desired tissue type. For example, spheroids derived from human fibroblasts are expected to express markers associated with human mesenchymal stem cells (MSCs). These markers include CD73, CD105, CD106, CD146, CD166, and STRO-1, which is well-established indicators of MSCs. Additionally, CD19 and CD45 are used as a negative marker, confirming the purity of the MSC population. Similarly, human hepatocyte-derived spheroids are expected to express human hepatic stem cell-specific surface markers such as CD117, CD133, and EPCAM. Spheroids derived from human keratinocytes are expected to display keratinocyte stem cell markers TfR / CD71 and p63 / TP73L. The expression of these markers is assessed using immunostaining techniques, for instance immunofluorescence staining, allowing for precise identification and confirmation of the stem cell populations. A person skilled in the art would understand that any similar staining technique can be employed here.

[0079] Differentiation into Target Tissue Cells: The tissue-specific adult stem cells, formed into spheroids, are subsequently cultured under defined conditions that promote their differentiation into cell types specific to the target tissue. For instance, in the case of generating human liver cells (hepatocytes), spheroids derived from human hepatocyte are cultured in a specialized medium tailored to support hepatocyte differentiation, including commercially available essential growth factors and signaling molecules, for instance, for human hepatocytes, Williams Medium E supplemented with HepExtend™ Supplement or Hepatocyte Maintenance Supplement Pack from ThermoFisher. Similarly, to generate human keratinocyte cells, keratinocyte-derived spheroids are cultured in a medium optimized for keratinocyte maturation, for instance EpiLife™ Medium, \OYN / * e? IGQIOZR RKJOZR LWTR :OHIT( 7ZWOSM YNK JOLLKWKSYOGYOTS UWTIKXX& YNK OJKSYOY^ TL YNK IKQQX is confirmed through the expression of tissue-specific proteins. For instance, CK19 serves as a marker of biliary differentiation during the differentiation of human hepatocyte-derived spheroids into liver duct cells, and this can be verified using techniques such as immunostaining. This step ensures that the cells not only acquire the appropriate morphology but also exhibit the functional markers characteristic of fully differentiated, tissue-specific cells.

[0080] Several other cell-types can be cultured and treated similarly to obtain tissue-specific adult stem cells. E.g. Generating human cartilage-producing cells (chondrocytes) from human skin fibroblasts: Human chondrocytes are specialized cells responsible for producing and maintaining cartilage, and they are typically differentiated from bone marrow-derived mesenchymal stem cells (MSCs). However, current regenerative medicine procedures aimed at cartilage regeneration,16 such as for treating knee degeneration, are invasive and rely heavily on the harvesting of MSCs from bone marrow. The current method is not only invasive but can also pose challenges in terms of scalability and patient recovery. Therefore, there is a clear need for the development of less invasive and more accessible techniques to produce cartilage-producing cells for regenerative treatments.

[0081] The generation of human chondrocytes from skin fibroblasts presents a promising alternative, utilizing the suspension culture method to provide a more accessible cell source. As described, fibroblast-derived spheroids can be created using this method, and these spheroids express surface markers characteristic of mesenchymal stem cells (MSCs). This approach offers a significant advantage, as fibroblasts are easier to obtain than bone marrow cells and can be reprogrammed to mimic MSC characteristics. Once these spheroids are formed, they can be treated with chondrogenesis differentiation media, such as the MesenCult™-ACF Chondrogenic Differentiation Kit from StemCell Technologies, guiding their differentiation into functional chondrocytes. This innovative process of transforming human skin fibroblasts into cartilageproducing chondrocytes using the suspension culture method offers a more feasible and patientfriendly alternative to current invasive techniques, marking a significant advancement in regenerative medicine. The accessibility of fibroblasts, combined with the ability to induce their differentiation into chondrocytes, provides a less invasive option for patients requiring cartilage regeneration. Additionally, the scalability of this approach makes it a promising candidate for widespread use in clinical settings, potentially overcoming many of the limitations associated with bone marrow-derived mesenchymal stem cells. The ease of obtaining fibroblasts and the efficiency of the suspension culture method suggest that this technique could be produced at scale, making it suitable for a broad range of therapeutic applications. With further refinement, this method has the potential to become a standard, minimally invasive procedure for treating cartilage-related conditions.

[0082] Figure 16 illustrates the procedure for generating human cartilage-producing cells (chondrocytes) from human skin fibroblasts. Fibroblast-derived spheroids exhibiting mesenchymal stem cell (MSC) characteristics are created using the suspension culture method. These spheroids are then cultured in a chondrogenesis differentiation medium to promote their differentiation into chondrocytes.17

[0083] Figure 17 shows Alcian blue staining of chondrocytes generated from human skin fibroblasts using the suspension culture method, with the chondrocytes staining positive for Alcian blue, in contrast to the human skin fibroblast control, which stained negative. A section of ribcage was used as a positive control.

[0084] Figure 18 illustrates the characterization of spheroid-derived chondrocytes generated using suspension culture method, through immunofluorescence staining, using specific markers for human chondrocytes, including Collagen type II (Coll type II), Aggrecan, and Cartilage Acidic Protein 1 (CRTAC1), which are indicative of chondrocyte identity. The staining results are presented for both human skin fibroblasts (top) and spheroid-derived chondrocytes (bottom), allowing for a comparison of marker expression between the two culture conditions. Technical discussion

[0085] This innovative approach addresses several key challenges associated with traditional reprogramming techniques. The technical foundation of this invention lies in the precise manipulation of cell signaling pathways, enabling the reprogramming of somatic cells into tissuespecific adult stem cells without the need for transgenes or viral vectors. By harnessing the inherent plasticity of somatic cells, particularly when they are detached from the culture substrate and placed into a specialized suspension culture environment, the invention guides these cells toward a stem-like state of their origin tissue. Therefore, this reprogramming is achieved solely through the modulation of cell signaling pathways in suspension culture environment and in the defined culture conditions, without the introduction of exogenous genetic material. Data obtained from single-cell RNA sequencing (scRNA-seq) using mouse skin fibroblast suggest that the following genes are targeted, and their expression is increased in the spheroids with mouse mesenchymal stem cell characteristics, derived from mouse skin fibroblasts: Notch2, Sox family (Sox4, Sox9), Foxp1, Klf2 and Hox genes, including Hoxb7, Hoxb9, Hoxb13 and Hoxa11os.

[0086] This innovative approach addresses several key challenges associated with traditional reprogramming techniques. First and foremost, by circumventing the use of genetic manipulation, the method dramatically reduces the risk of tumorigenesis. By avoiding use of viral vectors and transgenes, this method ensures a safer reprogramming process, preserving the genomic integrity of the resulting stem cells. In addition to its safety advantages, the method produces a significantly higher yield of tissue-specific adult stem cells compared to other approaches. This high efficiency makes it an ideal platform for clinical-scale production, providing sufficient18 quantities of stem cells for therapeutic applications such as regenerative medicine, disease modeling, and drug screening. Moreover, the method directly generates tissue-specific adult stem cells, bypassing the intermediate pluripotent stage that is typically required in conventional stem cell reprogramming. This not only simplifies the reprogramming process but also reduces the time and complexity involved in differentiation. As a result, the process is more streamlined and efficient, making it highly applicable for therapeutic use in regenerative medicine.

[0087] Adult stem cells (ASCs), also referred to as somatic or tissue-specific stem cells, are a population of undifferentiated cells that inhabit distinct anatomical locations within specific organs or tissues. These cells exhibit the remarkable capacity to differentiate into two or more distinct cell types, each possessing the unique characteristics and functionalities inherent to the tissue or organ of origin. This inherent multipotency of ASCs plays a pivotal role in tissue homeostasis, regeneration, and repair, making them indispensable players in the realm of regenerative medicine and biological research. ASCs are commonly isolated and maintained under conventional adherent culture conditions, where they represent an exceptionally scarce subpopulation of non-adherent cells. These cells possess the unique ability to aggregate and form spherical structures known as "floating spheres," which serve as a hallmark of their stem cell identity. These spheres exhibit distinctive stem cell characteristics, such as self-renewal potential and the capacity to differentiate into multiple cell lineages.

[0088] The inventor initially observed that when fibroblasts are cultivated in suspension culture, they undergo a notable transformation in morphology and concurrently exhibit the expression of stem cell-associated markers. However, this intriguing phenomenon is transient, as these cells subsequently enter a phase of rapid apoptosis (anoikis or detachment-induced cell death). Therefore, the inventor explored whether or not ASCs could be derived in larger numbers from suspensions of somatic cells cultured under non-adherent conditions.

[0089] The inventor initially studied mouse dermal fibroblasts (tail / ear fibroblasts, TEFs) in novel suspension culture conditions together with newly formulated growth factor (GF)-enriched, serumfree culture media with the Rho-kinase inhibitor, designed to support the transformation, survival and proliferation of ASCs. Materials and Methods

[0090] Preparation of Tail / Ear Fibroblasts (TEFs)19

[0091] To prepare tail / ear fibroblasts (TEFs), the tails and ears from adult mice (8–12 week-old C57BL / 6, FVB and Ly6a-GFP mice on a B6;129 background) were peeled, minced into 1 cm pieces, placed on culture dishes and incubated in Dulbecco’s Modified Eagle Medium / Nutrient Mixture F-12 (Gibco™ DMEM / F-12, HEPES, Cat#11-330-032, Thermo Fisher Scientific, Waltham, MA, USA), containing 1% Penicillin and Streptomycin (Cat#15140122, Thermo Fisher Scientific, Waltham, MA, USA) and 20% heat-inactivated fetal bovine serum (FBS; Cat# F1051- 500 mL, Sigma-Aldrich, Oakville, ON, Canada) for 7 days. Cells that migrated out of the graft pieces were transferred to new plates and maintained in DMEM / F-12 supplemented with 10% FBS (passage 2). TEFs from passage 3 were used for all experiments.

[0092] Suspension Culture Methods Causing Cell–Cell Contact

[0093] To overcome anoikis, cells were forced into physical contact using one of two methods that achieved similar results. (A) Hanging Drop Suspension Culture. In this method, the force of gravity encourages cell– cell contact. Cells were pretreated for one hour with the Rho-kinase inhibitor (ROCKi; Y-27632, 10 µM), then harvested via trypsinization and seeded at 100 cells per 25 µL drop of DMEM / F-12 supplemented with mouse or human EGF, mbFGF and mIGF-1 (all 20 ng / mL) for TEFs and HDFs, respectively, plus 10 µM Y-27632 on the inner side of a 100 mm tissue culture Petri dish lid. For PHHs, Williams Medium E serum-free medium containing hHGF, bFGF and hIGF-1 (all 20 ng / mL) and ROCKi (10 µM) was used, and for human keratinocytes, SFM serum-free medium supplemented with hEGF, bFGF and hIGF-1 (all 20 ng / mL) and ROCKi (10 µM) was used. The lid was carefully turned upside down (hanging drop) and placed on top of a Petri dish filled with 5 mL of sterile PBS and the cells were cultured in an inverted position in separate drops for 2 days. The hanging drops were maintained in a humidified atmosphere of 5% CO2 at 37 °C. Cells were monitored under a fluorescence microscope daily to detect the creation of aggregates. Images were taken using EVOS FL Auto Cell Imaging System (Thermo Fisher Scientific, Waltham, MA, USA). On day 3, aggregated cells were transferred to ultra-low-attachment (ULA) 96-well plates (Corning® Costar® Ultra-Low Attachment Multiple Well Plate, Cat#7007) containing 200 µL of the same medium in each well. Cells were maintained in 5% CO2, 21% O2 and 74% N2 at 37 °C for 7 days. Half of the culture medium was removed and replaced every other day. (B) G-force via Centrifugation (250× g for 3 min). Cells were plated at 100–300 cells per well in ULA 96-well round-bottom plates (Corning® Costar® Ultra-Low Attachment Multiple Well Plate,20 Cat#7007) in 200 µL of DMEM / F-12 supplemented with mouse or human EGF, mbFGF and mIGF-1 (all 20 ng / mL) for TEFs and HDFs, respectively, plus 10 µM ROCKi on the inner side of a 100 mm tissue culture Petri dish lid. For PHHs, Williams Medium E serum-free medium containing hHGF, bFGF and hIGF-1 (all 20 ng / mL) and ROCKi (10 µM) was used, and for human keratinocytes, SFM serum-free medium supplemented with hEGF, bFGF and hIGF-1 (all 20 ng / mL) and ROCKi (10 µM) was used. Cells were maintained in 5% CO2, 21% O2 and 74% N2 at 37 °C for 7 days. Half of the culture medium was removed and replaced every other day.

[0094] Time Lapse Image Acquisition of Sphere Growth

[0095] The growth of spheres was imaged (phase-contrast and / or green fluorescence) every 60 min using an IncuCyte® live cell analysis system (Essen BioScience, Ltd., Royston Hertfordshire, UK) equipped with a 20× objective over a 7-day time course.

[0096] Flow Cytometry

[0097] For the apoptosis assay, apoptotic cells were quantified using an Annexin V (Alexa Fluor™ 555 conjugate, Cat# A35108, Thermo Fisher Scientific, Waltham, MA, USA), in accordance with the manufacturer’s protocol. Briefly, at the desired time point, both lifted adherent cells or cells in suspension medium were collected via centrifugation at 250 RCF at 4 °C for 5 min, followed by two cold 1 × PBS washes. Cells were then resuspended in binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl and 2.5 mM CaCl"% GY G ITSIKSYWGYOTS TL + c +*g IKQQX)R>( 5WOKLQ^& +** e> TL YNK IKQQ XZXUKSXOTS \GX YWGSXLKWWKJ YT G . R> IZQYZWK YZHK& GSJ . e> TL 4SSK]OS V (555) was added. Unstained cells were used as a negative control. The cells were gently [TWYK]KJ GSJ OSIZHGYKJ OS YNK JGWP LTW +. ROS GY WTTR YKRUKWGYZWK( 4S GJJOYOTSGQ ,** e> TL HOSJOSM buffer was added to the cell suspension and Cells were analyzed using BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lakes, NJ, USA) to quantify percentages.

[0098] Immunophenotype Analysis Using Flow Cytometry: Antibodies used for the immunophenotype analysis of mMSC and hMSC surface markers are listed in Table 3. Cell subsets were stained with antibodies and the isotype control and analyzed using BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lakes, NJ, USA). Collected events per sample numbered 15,000. Data were analyzed using FlowJo software.

[0099] Immunofluorescence (IF) Staining21

[0100] Immunofluorescence was performed as described previously. Samples (cells and spheroids) were washed with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. After washing twice with PBS, cells were permeabilized and blocked in a solution of 1 × PBS containing 0.1–0.5% Triton X-100 and 1% Carbo-Free Blocking Solution (SP-5040-125, Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. Samples were then incubated with desired primary antibodies at 4 °C overnight (the list of antibodies and concentrations used can be found in Table 3). After washing three times with PBS, secondary antibodies (1:200, Jackson ImmunoResearch, West Grove, PA, USA) were incubated at 37 °C for 1 h at room temperature in the dark. Nuclei were stained with DAPI (Roche Life Science) for 15 min. Images were digitally captured using an epifluorescent microscope (Zeiss Axioimager M2 with Apotome 2, Carl Zeiss Microscopy GmbH, Göttingen, Germany) using appropriate filter sets. Identical images acquired with different filter sets were merged using ZEN Pro software (version 2.6, Carl Zeiss Microscopy GmbH).

[0101] Table 3: Resources222324

[0102] Self-Renewal Assay

[0103] To assess whether or not cells within TEF-derived spheroids could be propagated as secondary cultures to determine their self-renewal capacity, the inventor used an established method with slight modifications. Spheres were transferred into a small tube and after neutralizing trypsin with media, spheres were mechanically broken down by pipetting the solution up and down with a small pipet tip. Cells were counted using a hemocytometer and trypan blue, single cells were then seeded in 96-well culture plates with 200 µL of DMEM / F-12 in each well, supplemented with GFs with a limiting dilution approach and wells containing a single cell were monitored for proliferation and clonal expansion. The frequency of expansion from single cells was calculated by dividing the number of wells containing new spheroids by the total number of wells containing a single cell.

[0104] In Vitro Differentiation Assays of Mouse Adipogenic or Osteogenic Differentiation

[0105] Fibroblasts and MSC-derived spheres were plated on a 2-well chamber slide and grown in 10% DMEM / F-12. Next day, the medium was changed to adipogenic or osteogenic differentiation media (StemXVivo steogenic / Adipogenic Base Media, cat# CCM007, RD systems, Minneapolis, MN, USA) and replaced every 3–4 days for a total of 21 days. Adipocyte lipid droplets and osteoblast calcification were detected via oil red O and alizarin red S staining, respectively.

[0106] Single-Cell RNA Library Preparation and Sequencing

[0107] SIST cells were prepared in accordance with the protocol described in this study. Between 70,000 and 100,000 mouse adherence-cultured fibroblasts (monolayer) and dissociated spheroid cells (single cells) were freshly prepared and their gene expression profile analyzed using singleIKQQ -d C@4'XKVZKSIOSM( DKVZKSIOSM QOHWGWOKX \KWK UWKUGWKJ ZXOSM DOSMQK 6KQQ -d CKGMKSY =OYX V3.1 (10x Genomics, Pleasanton, CA, USA) with the 10x Chromium controller, and sequenced on NextSeq 500 (Illumina, San Diego, CA, USA).

[0108] Bioinformatics Analysis

[0109] Sequencing data were processed with Cellranger v7Ø0 to generate cell vs. gene Unique Molecular Identifier (UMI) count matrices using the 10x Genomics mouse genome reference (refdata-gex-mm10-2020-A). Matrices were loaded into R (v4.2.1) and subsequent analysis was25 performed in Seurat v4.3Ø Data from the attached and suspension culture libraries were separately run through a series of quality control steps, retaining cells with at least 200 detected genes and including only genes detected in more than three cells. The data from each library were processed with scDblFinder v1.10.0 using default parameters to identify potential cell doublets, and each library was filtered to remove doublets and cells with >25% mitochondrial transcripts. UMI counts in the retained cells were normalized using the SCTransform algorithm and the libraries were integrated using the Seurat data integration pipeline with 3000 features. Principal component analysis (PCA) was run on the integrated assay, a nearest neighbor graph was constructed using the first 25 principal components and cells were clustered using the Louvain algorithm at a resolution of 0.3. Uniform manifold approximation and projection (UMAP) embedding was performed for data visualization, again using the first 25 principal components (integrated UMAP). To visualize differences between the two libraries, SCTransform was run on the raw count data from the combined libraries, followed by PCA and the conduction of a second round of UMAP embedding using the first 25 principal components of this new PCA dimensional reduction (SCTransform UMAP). Markers for each cluster were identified using the Seurat FindAllMarkers function with the Wilcox test, searching only for markers with a positive log2 fold change. To identify differentially expressed genes between pairs of clusters or between suspension and attached cells, the FindMarkers command was used.

[0110] Human Hepatocyte Differentiation

[0111] To assess the capacity of differentiation between PHH-derived spheroids and ductal cells, spheres were seeded in a serum-free medium, Williams Medium E, containing hHGF, bFGF and hIGF-1 (all 20 ng / mL) until they formed a monolayer. The medium was then changed to a transition and expansion medium (TEM) containing DMEM / F12 supplemented with insulintransferrin-serine (ITS) (Cat# I3146, Sigma-Aldrich, Oakville, ON, Canada), with the following growth factors or small molecules: hEGF (20 ng / mL, Cat# 78006.1, Stemcell technologies, Vancouver, BC, Canada), hHGF (20 ng / mL, Cat# 100-39H, Peprotech, Cranbury, NJ, USA), F,0 / -, $+* e?& 6GY" +***..1-%& 6;<C22*,+ $- e?& 6GY" +-+,,'+%& DUNOSMTXOSK'+'UNTXUNGYK $D+B% $+ e?& 6GY" / ,.0*%& Q^XTUNTXUNGYOJOI GIOJ $>B4% $. e?& 6GY"+**+**2-'+% GSJ 41-'*+ $+ e?& 6GY" 2**+022'.% $GQQ LWTR 6G^RGS 6NKROIGQ& 4SS 4WHTW& ?<& ED4%& LTW 0 JG^X GX JKXIWOHKJ previously.

[0112] Tumorigenic Assessment of TEF-Derived Spheroids26

[0113] Mouse fibroblast cells and spheres were suspended at 1 × 107 cells / mL in DMEM / F12 ITSYGOSOSM +*# 95D( 54>5)I SZJK ROIK \KWK GSKXYNKYO_KJ GSJ +** e> TL YNK IKQQ XZXUKSXOTS $+ × 106 cells) was injected subcutaneously into the dorsal flank. Ninety days after the injection, tissues of the dorsal flank were dissected from the mice, fixed in 4% formaldehyde and embedded in paraffin. Sections underwent hematoxylin and eosin staining and were evaluated.

[0114] Statistics

[0115] All numerical data are presented as mean ± SEM from at least three separate experiments. p-values were obtained via a 2-tailed t-test for 2 groups, or a one-way analysis of variance (ANOVA) followed by a post hoc Tukey test for more than 2 groups using GraphPad Prism Version 6.0 software (GraphPad Software, San Diego, CA, USA). Differences were considered significant at p < 0.05. Results

[0116] Expression of MSC Surface Markers by Mouse TEFs Grown in Suspension Culture While Undergoing Detachment-Induced Apoptosis (Anoikis)

[0117] Stem cell antigen-1 (Sca1; Ly6A) is a well-established marker of murine hematopoietic and mesenchymal stem cells (MSCs). The inventor used dermal fibroblasts containing a Ly-6A (Sca-1) GFP (Sca-1-GFP) transgene to monitor the conversion of TEFs into MSCs. TEFs from 8- to 12-week-old Sca-1-GFP transgene mice were isolated and plated (500 cells / well) on ultra-lowattachment (ULA) 96-well plates. Once detached, the elongated and flattened cells transformed into a rounded morphology with large nuclei and a scant cytoplasm (Figure 1A). Within 24h, an increase in GFP expression (endogenous Sca-1) was detected (Figure 1B–E). GFP expression was rapidly lost when cells were moved back to the adherent culture (Figure 7A), suggesting the possible conversion of TEFs into MSC-like cells in the suspension. Consistent with this interpretation, flow cytometric analysis using mMSCs surface markers Sca-1, CD29, CD44, CD90.1, CD105, and CD106, and CD45R as a negative marker, revealed a transition of wild-type TEFs after 24 h of suspension culture to an MSC-like phenotype (Figure 1F). However, the majority of MSC-like cells derived using this method, which the inventor termed as suspension-induced stem cell transition (SIST), subsequently underwent apoptosis (anoikis), as evidenced by the increased expression of Annexin V (Figure 1G,H) and cleaved Caspase 3 (Figure 7B).

[0118] Figure 1. Mouse TEFs grown in suspension culture display MSC surface markers while undergoing detachment-induced apoptosis (anoikis). (A) Comparison of Sca-1-GFP TEFs27 morphology as a monolayer (top, pointed by white and black arrows) and after 30 min in suspension (bottom, pointed by white and black arrows). GFP fluorescence images are combined with their corresponding phase-contrast images. (B) Flow cytometric analysis of GFP expression of adherent wild-type or Sca-1-GFP TEFs cultured as a monolayer and cells cultured in suspension. (C) Quantitative evaluation of GFP expression of adherent Sca-1-GFP TEFs from (B). (D) Representative immunoblots of GFP protein in Sca-1-GFP TEFs grown as a monolayer and 24 h in suspension. (E) Densitometric analysis of GFP protein expression in monolayer Sca- 1-GFP TEFs vs. suspension (n = 3). (F) Representative histograms for flow cytometric analysis of wild-type mouse TEFs cultured as a monolayer and 24 h in suspension. TEFs were analyzed for mMSCs surface markers, Sca-1, CD29, CD44, CD90.1, CD105, CD106 and CD45R of adherent (top) and suspension cells after 24 h (bottom). (G) Flow cytometry analysis for detection of Annexin V (Alexa 555) of TEFs grown for 14 h in adherent or suspension conditions. (H) Quantification of Annexin V-positive cells from (F). *** p < 0.001.

[0119] Figure 7 shows mouse TEFs grown in suspension culture and expressing stem cell markers undergo cell death. (A) Co-immunofluorescence staining of Sca-1 (red) with apoptosis marker cleaved Caspase 3 (C.CASP3) (green) in TEFs grown for 24 h in adherent (top) and suspension culture (bottom). Representative images of spheres of TEFs isolated from (B) C57 (top) and (C) FVB (bottom) wild-type mice and quantification of their spheroids size at day-7 compared to day one. (mean ± SEM, n = 8 spheroids). Scale bars are indicated in the images. ***p<0.001. Scale bars: 50 µm.

[0120] Overcoming Anoikis with Generation of Proliferative Mouse TEF-Derived Spheroids Expressing MSC Markers with Self-Renewal Capacity

[0121] It is known that pro-survival signaling pathways are activated via cell–cell and cell-matrix anchorage and that detachment triggers anoikis. The inventor therefore examined whether or not a modified suspension culture, designed to favor cell–cell contact, may avoid anoikis. TEFs in suspension were brought into physical contact either via gravity, using hanging drop suspension culture (Figure 2Ai), or via centrifugation (250× g for 3 min) in ULA 96-well round-bottom plates (Figure 2Aii). The increase in GFP expression observed within 24 h in spheroids generated using both methods confirmed the activation of endogenous Sca-1, (Figure 2B). For all subsequent experiments, ULA 96-well plates were employed, as they were more suitable for live cell imaging.28

[0122] Figure 2. Mouse TEF-derived spheroids grown in SIST express mMSC markers, proliferate and exhibit self-renewal capacity. (A) Schematic representation of two suspension culture methods designed to promote cell–cell contact. (B) Representative microscope images captured from live cell imaging from Sca-1-GFP fibroblasts grown in suspension culture (left) and sphere formation (right) at 24 h. (C) IF staining of cleaved caspase 3 (C.CASP3) of mouse TEFs treated with 0.4 mM hydrogen peroxide as positive control (top) and mouse TEFs spheroids after 7 days (bottom). (D) IF staining of mouse MSCs-specific surface markers Sca-1, CD29, CD44, CD90.1, CD105, CD106 and CD45R as a negative marker of adherent cells (top) and spheroids (bottom). (E) Quantification of spheroid volume over a 7-day time course (n = 6 spheroids). (F) Schematic representation of methods used for self-renewal evaluation of mouse fibroblastderived spheroids in this study. (G) Representative 96-well plate stained with crystal violet to identify wells with new spheroids. *** p < 0.001.

[0123] To characterize wild-type TEF-derived spheroids, they were initially transferred to an 8- well chamber slide and allowed to adhere for 1 h. The inventor first evaluated whether or not the formation of spheroids using the SIST method reduces anoikis induced via detachment by conducting IF staining using an antibody specific for cleaved caspase 3 (C.CASPASE3). The IF staining for C.CASPASE3 demonstrated the absence of apoptotic cells within the spheroids (Figure 2C). The inventor then assessed the expression of mMSC surface markers Sca-1, CD29, CD44, CD90.1, CD105, CD106 and CD45R as a negative marker via IF. As shown in Figure 2D, the majority of cells were positive for MSC markers and negative for CD45R. To evaluate proliferation capacity, spheroid volume was measured over 7 days; steady growth was observed (Figure 2E). To ensure the observations in TEFs were not mouse strain-dependent, spheroid formation was reproduced using TEFs from C57BL / 6J and FVB / NJ wild-type murine strains (Figure 7C,D). To further examine the proliferative capacity of spheroid cells were stained for the proliferation marker Ki-67 (Figure 8A). The proliferative index (proportion of Ki67 positive cells) was significantly increased in spheroids compared to adherent TEFs (Figure 8B). Therefore, TEFderived spheroid cells using the novel SIST platform remain viable, are highly proliferative and express MSC markers.

[0124] Figure 8 shows Mouse TEF-derived spheroids are highly proliferative. (A) Representative IF staining for Ki67 expression in mouse fibroblasts grown in monolayer (2D) and spheroids grown in suspension culture (3D). Spheroids cultured for 7 days were stained for the presence of the proliferation marker Ki-67 (anti-Ki-67) and nuclei were stained with DAPI. (B) Quantitative analysis29 of Ki67-positive as a % of total cells. (mean ± SEM, n = 7-8 spheroids). Scale bars are indicated in the images. ***p<0.001.

[0125] The inventor next examined the self-renewal capacity of wild-type TEF-derived spheroid cells, using a well-established assay. Spheroids were enzymatically and mechanically dissociated, and cells were seeded, one per well, in 96-well plates (288 wells total). The formation of clones was then evaluated after 14 days (Figure 2F). As shown in Figure 2G, a significant increase in the number of new colonies generated from spheroid cells (9.89%) compared with adherent cells (0.78%) was observed. These observations suggest that SIST spheroids comprise a heterogeneous population of MSCs and progenitor cells without self-renewal capacity. The inventor next examined whether or not SIST-derived spheroid cells are multipotent. Individual spheres were transferred using a micropipette onto a 2-well chamber slide. After plating and sphere adherence to the slide, cells were observed to migrate away, causing the gradual loss of the three-dimensional structure over a 5-day period (Figure 3A). Double IF staining revealed that migrating cells lost their immunoreactivity for Sca-1 and CD44 (Figure 3B), similar to the loss of GFP expression in Sca-1-GFP spheroids in adherent culture (Figure 3A, bottom). After two days in culture and forming a monolayer, cells were placed either in osteogenic or adipogenic induction culture media for 21 days. Consistent with the degree of differentiation, adipocyte lipid droplets and osteoblast calcification were detected using Oil Red O and Alizarin red S, respectively (Figure 3C). To assess tumorigenic potential, TEFs (control) and spheroid cells were injected into the dorsal flanks of BALB / c nude mice and monitored over three months. No tumors were observed in any mice, which was confirmed via histological examination (Figure 3D).

[0126] Figure 3. Mouse TEF-derived spheroids are multipotent with no tumorigenic potential. (A) Seeding of a single sphere of wild-type (top) and Sca-1-GFP (bottom) fibroblasts on a coverslip after 1 and 5 days in culture. (B) Co-IF staining of Sca-1 (red) with another marker of MSCs CD44 (green) in a single sphere after 1 (left panel) and 5 days (right panel) in culture. Scale bars are indicated in the images. (C) Representative images of Oil Red O (left) and Alizarin Red S (right) staining demonstrating adipogenic and osteogenic differentiation of adherent fibroblasts (middle panel) from TEFs derived from spheroids (bottom panel) and adherent fibroblasts (left panel). Mouse liver and bone tissue were used as positive controls (top panel). (D) Representative hematoxylin and eosin staining of tissues dissected from injection sites in mice receiving cell-free vehicle (Vehicle), adherent monolayer fibroblasts or spheroid-derived cells (Spheroids).30

[0127] Characterizing the Global Gene Expression Profile of Mouse TEF-Derived Spheroids Compared to Monolayer Culture

[0128] To identify the transcriptional changes resulting from growth in spheroid vs. monolayer conditions, the inventor performed single-cell RNA sequencing (scRNA-seq) on cells grown under both conditions. After performing quality control steps to remove presumed low-viability cells (>25% mitochondrial transcripts) and cell doublets, 11,515 cells in total (8022 from the spheroid culture, and 3493 from the monolayer culture) were retained for analysis. Data from the two libraries were combined using the Seurat integration pipeline to group similar cells from both libraries and the data were visualized using uniform manifold approximation and projection (UMAP). A nearest-neighbor graph was constructed using the integrated assay and clustering was performed using the Louvain algorithm at a resolution of 0.3, identifying eight clusters (Figure 4A). Coloring cells by source library in the UMAP projection shows that the integration procedure intermixed cells from the two libraries in the UMAP space, in contrast to the UMAP projection generated on the non-integrated data, in which the cells from the two libraries are separate in UMAP space (Figure 9A). The cells of both libraries can also be split into populations with a high $3,.**% TW QT\ $a,.**% SZRHKW TL JKYKIYKJ MKSKX& \OYN GS OSIWKGXKJ SZRHKW TL QT\KW MKSK ITZSY cells from the spheroid culture (Figure 9B).The low- and high-gene cells group separately in the UMAP space and Louvain clustering, with clusters 0, 1, 2 and 6 containing the low gene cells, and clusters 3, 4, 5 and 7 containing high-gene cells. The identification of marker genes for each cluster revealed between 233 and 1671 markers per cluster (with an adjusted p-value of < 0.05), with 3365 genes in total being identified as markers of at least one cluster. In these clusters, spheroid cells comprise the majority of clusters 0, 1, 2, 4 and 7 (Figure 9C). The heatmap suggested differential gene expression across all clusters (Figure 9D), which supports their separate distribution in UMAP (Figure 4B). As seen in Figure 9D, clusters 1 and 6 group together in the top dendrogram, as do clusters 4 and 7. The analysis of differentially expressed genes (DEGs) revealed that each cluster was characterized by a specific transcriptional profile.

[0129] Figure 4. Characterization of mouse TEF-derived spheroids vs. monolayer via differential expression gene analysis. (A) UMAP projection of cells calculated from a principal component analysis (PCA) reduction from the Seurat integrated assay, which attempts to bring similar cells close together. Cells are colored by (A) cluster, identified from the integrated assay at a resolution of 0.3, or by (B) source library, showing the overlap of UMAP coordinates. (C) Violin plots of MSC surface marker genes observed in IF staining in Figure 2C, showing expression profiles split by source library within each cluster. (D) Pie chart demonstrating distribution of spheroid cells across31 clusters. (E) Violin plot visualization of expression of MSC surface markers genes identified as significantly enriched in cluster 4. (F) Violin plot visualization of expression of selected collagen, fibronectin and laminin genes identified as significantly enriched in cluster 4. (G) Violin plots of stem cell self-renewal genes identified as significantly enriched in cluster 4. (H) Violin plot visualization of expression of homeobox genes identified as significantly enriched in cluster 7.

[0130] Figure 9. Transcriptome analysis using RNA-seq in TEFs cultured as a monolayer and spheroids. (A) (A) UMAP projection of cells from the attached (monolayer) and suspension (spheroid) cells. UMAP embeddings calculated from a PCA reduction of the SCTransformed UMI counts, showing that the cells from the two libraries have generally nonoverlapping UMAP embeddings. (B) Violin plots showing distributions of the number of detected genes (nFeature_RNA) and number of UMIs (nCount_RNA) per cell in the two libraries. Cells fall into two populations, with ‘high’ (> ~2,500) or ‘low’ (< ~2,500) numbers of detected genes. The absolute number of ‘high’ gene cells is similar in the two libraries, but the Spheroid library has a large excess of ‘low’ gene cells. (B) (C) Bar plot showing the number of cells from each library in each of the resolution 0.3 clusters identified on the integrated assay. (D) Heatmap illustrating differentially expressed genes (DEGs) of mouse TEFs cultured as a monolayer and spheroids in across 8 clusters, illustrating that each cluster exhibited unique gene expression.

[0131] Figure 10. Expression distribution for selected mouse MSCs-specific genes. (A) UMAP projections (integrated assay) showing the distribution of UMI counts, and the five MSC surface markers genes observed in IF staining in Figure 2D across the dataset (negative for CD45 (Ptprc) gene).

[0132] Next, the inventor examined the expression of the MSC surface markers observed in IF staining (Figure 2C) in each cluster. Consistent with the IF results in Figure 2C, the expression levels of mouse MSC genes, Sca-1 (Ly6a), CD29 (Itgb1), CD44 and CD90.1 (Thy1) were high in clusters 2, 3, 4, 5 and 7 (Figure 4C and Figure 10). During the initial clustering analysis conducted at a lower resolution, cluster 4 and cluster 7 were grouped together into a single cluster. However, upon a further refinement of the analysis at a higher resolution, they emerged as distinct individual clusters. Subsequently, guided by their distinct gene expression profiles, the inventor directed the32 focus towards conducting an in-depth analysis of clusters 4 and 7. The gene expression analyses revealed that cluster 4, which includes 9% of the 8022 original spheroid cells (Figure 4D), exhibited the significantly increased expression of MSC markers compared to the rest of the cells (Figure 4E). The inventor observed that the expression of many extracellular matrix (ECM) genes, including Col1a1, Col1a2, Col3a1, Fbn1, Lama2 and Lama4, was significantly greater in cells of one of more clusters among clusters 2, 3, 4, 5 and 7 (Table 1 and Figure 11A,B), and that expression of some of these genes was significantly higher in spheroid cells than in attached cells. Further, their expression was significantly increased in spheroid cells of cluster 4 compared with the rest of the cells (Figure 4F). Since approximately 10% of spheroids cells showed a selfrenewal property (Figure 2F), the inventor sought to explore the genes important for stem cell self-renewal. The analysis of differentially expressed genes revealed the significantly increased expression of one or more of the genes Notch2, Sox4, Sox9, Klf2 and Foxp1 in clusters 3, 4, 5 and 7 (Table 2 and Figure 12A), but only in cluster 4 was the expression all of these genes significantly greater when compared to that of the remainder of the dataset, with that of Sox9 and Foxp1 also being significantly higher in spheroid vs. monolayer cells within cluster 4 (Table 2 and Figure 4G and Figure 12B). The heatmap shown in Figure 12C displays the expression profile of selected self-renewal and collagen genes across clusters.

[0133] Table 1: ECM Genes

[0134] All of the six selected genes (Col3a1, Col1a2, Col1a1, Fbn1, Lama4, Lama2) are significantly higher in cluster 4 than in the remainder of the dataset. Col1a2 is significantly higher in all of cluster 2, 3, 4, 5, and 7.

[0135] Table 2: Self Renewal Genes33

[0136] The self-renewal genes (Foxp1, Sox9, Sox4, Fbn1, Notch2, Klf2), are significantly higher in cluster 4 than in the remainder of the dataset.

[0137] Figure 11. Expression distribution of selected ECM genes in monolayer and spheroid cells. (A) Violin plot illustrating expression of the six selected ECM genes in monolayer and spheroid cells across eight clusters. (B) Feature plots of expression distribution for six selected ECM genes with the lowest p-value in in the dataset. Expression levels for each cell are color-coded and overlaid onto the UMAP plot.

[0138] Figure 12. Expression distribution for self-renewal genes in monolayer and spheroid cells. (A) Violin plot showing expression of self-renewal genes Notch2, Sox4, Sox9, Klf2, and Foxp1 across eight clusters. (B) Feature plots of expression distribution of self-renewal genes Notch2, Sox4, Sox9, Klf2, and Foxp1. Expression levels for each cell are color-coded and overlaid onto the UMAP plot. (C) Heatmap shows the average expression level of all collagen genes present in the dataset and selected self-renewal genes across clusters.

[0139] The DEGs analysis of cluster 7, which had the lowest percentage of spheroid cells among the clusters (Figure 4D), showed the significant upregulation of a family of transcription factors, Hox genes, including Hoxb7, Hoxb9, Hoxb13 and Hoxa11os (Figure 4H and Figure 13A), and Mmp10 and Mmp13 genes (Figure 13B,C). Hox genes play a crucial role as regulators of periosteal stem cell identity, affecting fibroblast, osteoblast and progenitor cells. Additionally, Mmp genes are implicated in extracellular matrix (ECM) remodeling, a pivotal component of the stem cell niche. The heatmaps demonstrating average normalized expression per cluster of genes expressed at a significantly higher level in cluster 7 and all the Hox and Mmp genes in the dataset are presented in Figure 14A,B. Altogether, the characterization of transcriptional profiles of the34 TEFs-derived spheroid cells using scRNA sequencing revealed the presence of specific clusters associated with mesenchymal stem-like cells and self-renewing capacity.

[0140] Figure 13. Feature plots of expression distribution for Hox and Mmp genes. (A) UMAP visualization of expression distribution of selected Hox genes illustrating their higher expression in cluster 7. (B) Violin plot and (C) UMAP showing expression of Mmp13 and Mmp10 genes expressed more highly in cluster 7.

[0141] Figure 14. Hox and Mmp genes are highly expressed in cluster 7. (A) Heatmaps showing average normalized expression per cluster of genes expressed at a significantly higher level (adjusted p-value < 0.05) in cluster 7 than in all other cells in the dataset, in which clusters 4 and 7 look much more similar to each other; and (B) All Hox and Mmp genes with measured expression in the dataset. Normalized counts per cell were averaged for each cluster, and values were scaled and centered row-wise to plot Z-scores for each gene; rows and columns were hierarchically clustered using complete linkage clustering with a Euclidian distance metric. Sidebars in (B) show the log2 average normalized expression of each gene across the whole dataset and specifically in the cells in cluster 7.

[0142] Generation of Spheroids with SC Properties from Cells of All Three Human Germ Layers, Mesoderm, Endoderm and Ectoderm (Human Dermal Fibroblasts, Hepatocytes and Keratinocytes, Respectively)

[0143] The inventor next assayed SIST on primary human dermal fibroblasts (HDFs), which revealed a transition of HDFs after 24h suspension culture to a MSC-like phenotype as made evident via flow cytometric analysis using hMSCs surface markers: CD73, CD105, CD106, CD146, CD166, STRO-1 and CD45 (negative marker) (Figure 5A). HDFs also showed a capacity to form a growing spheroid similar to that of mouse TEFs (Figure 5B and Figure 15A). Stem cell characteristics were confirmed via IF staining using human MSC-specific surface markers CD105, CD73, CD106, CD146, CD166, STRO-1 and CD19 and CD45 (negative marker), (Figure 5C). Furthermore, similar to the case of TEFs, MSC markers were lost when spheroids were transformed to an adherent monolayer (Figure 15B,C).

[0144] Figure 5. Human dermal fibroblast- and hepatocyte-derived spheroids possess SC properties. (A) Representative phase-contrast images of adherent monolayer human dermal fibroblasts (top) and a spheroid after 7 days in culture (bottom). (B) Representative histograms35 for flow cytometric analysis of primary HDFs cultured as a monolayer and 24 h in suspension. HDFs were analyzed for surface hMSC surface markers CD73, CD105, CD106, CD146, CD166, STRO-1 and CD45 as a negative marker of adherent (top) and suspension cells after 24 h (bottom). (C) IF staining of human MSC-specific surface markers CD73, CD105, CD106, CD146, CD166, STRO-1 and CD45 as a negative marker of adherent cells (top) and spheroids (bottom). (D) Representative phase contrast images of adherent monolayer human hepatocytes (top) and a spheroid after 7 days in culture (bottom). (E) Characterization of human hepatocyte-derived spheroids via IF staining using human hepatic stem cell-specific surface markers CD117, CD133, EPCAM, and AFP and ALB as a hepatocyte marker on adherent cells (top) and spheroids (bottom). Sections from human liver tissue were used as positive control (top panel). Scale bars are indicated in the images.

[0145] Figure 15. Human dermal fibroblast-derived spheroids are proliferative. (A) Quantification of spheroid volume using real-time cell imaging by IncuCyte® live-cell analysis system over a 7- day time course (n = 6). (B) A single sphere of human fibroblasts after seeding on a coverslip after 1 (left) and 5 days (right) in culture. (C) Co- immunofluorescence staining of CD105 (red) with another marker of human MSCs CD166 (green) in a single sphere after 1 (left panel) and 5 days in culture (right panel).

[0146] Finally, the inventor examined whether or not other human somatic cells of endodermal (hepatocyte) or ectodermal (keratinocyte) origin could also undergo SIST. Primary human hepatocytes (PHHs) formed spheres (Figure 5D) that were less compact compared to the spheroids derived from TEF and HDF-derived MSCs and displayed a delicate and fragile nature. Consequently, a notable proportion of these spheroids partially disaggregated during the transfer process onto slides for further examination, including for immunofluorescence (IF) staining. Notably, despite some disaggregation, these spheroids still expressed human hepatic stem cellspecific surface markers CD117, CD133 and EPCAM, while testing negative for the hepatocyte marker AFP (Figure 5E). As for mesodermal cells, stem cell markers were lost when PHH-derived spheroids formed an adherent monolayer (Figure 6A,B). To confirm that PHH-derived spheroid cells are capable of differentiation into ductal cells, spheroids were grown to form a monolayer using an established transition and expansion medium for 7 days and then were showed to stain for CK19 (Figure 6C), a specific ductal cell marker. Primary human keratinocytes also generated proliferating spheres (Figure 6D) which were positive for stem cell markers TfR / CD71 and p63 / TP73L, and negative for keratinocyte markers CK1 and CK5 and CK14 (Figure 6E).36 Collectively, the results confirm the transformation of mouse and primary human somatic cells (originated from all three germ layers) into ASC-like cells when subjected to unique SIST suspension culture conditions designed to avoid anoikis.

[0147] Figure 6. Differential capacity of human hepatocyte-derived spheroids and formation of spheroids by human keratinocytes that proliferate and express keratinocyte-specific stem cell markers. (A) A single sphere of human hepatocytes after seeding on coverslip at day 1 (left) and day 5 (right) in culture. (B) Co-IF staining of CD117 (red) with AFP (green) in a single sphere after 3 days in culture. (C) IF staining of AFP (green) and CK19 (red, ductal cell marker) on human hepatocytes derived from spheroids after 7 days culture in transition / expansion medium. (D) Representative phase-contrast images of monolayer human keratinocytes (top) and a spheroid at day 7 (bottom). (E) IF staining of human keratinocytes CK1, CK5 and CK14 and keratinocyte stem cell markers TfR / CD71 and p63 / TP73L on adherent cells and spheroids. Scale bars are indicated in the images.

[0148] Herein, the inventor reported a novel suspension culture method that circumvents anoikis, thus allowing a comparatively large proportion of mouse and human somatic cells to form spheroids enriched with cells that have ASC-like properties. The inventor believes the conditions that favor the growth of ASCs in a non-adherent culture environment resulted from increased cell– cell contact in conjunction with optimized culture media. The mechanisms via which normal cells in non-adherent culture acquire ASC characteristics remain unclear. Most primary cells are considered to be anchorage-dependent for survival with anoikis rapidly ensuing once they are detached, complicating the examination of their properties in non-adherent culture. In contrast, adherence to a plastic substrate leads to the induction of a transcriptional and surface marker shift allowing for survival. Unlike normal cells, transformed or tumorigenic epithelial cells can proliferate when non-adherent, allowing an epithelial–mesenchymal transition (EMT) with the emergence of cells with stem cell properties. In contrast, the suspension culture method, herein termed SIST, encourages physical contact between cells to allow normal cells to avoid anoikis, thereby more efficiently forming spheroids containing ASC-like cells.

[0149] One of the first morphological changes the inventor observed in cells grown in suspension culture was their transformation into clusters of round-shaped cells with large nuclei and a scant cytoplasm, possibly reflecting a mesenchymal to epithelial transition (MET). Recent studies have shown that the generation of induced pluripotent stem cells from mouse fibroblasts requires the37 activation of intracellular MET signals, suggesting a cooperative process between exogenous transcription factors and the extracellular micro-environment. In accordance with this, the scRNAseq analysis revealed cellular and transcriptional modules associated with the ECM, MScs and stem cell self-renewal. Therefore, the observations raise the possibility that somatic cells are intrinsically capable of transforming into ASCs in non-adherent culture conditions by activating signaling pathways similar to those observed in MET.

[0150] Importantly, ASC-like cells generated using SIST appear to have normal a morphology while lacking tumorigenicity. Furthermore, using this strategy, the derivation of ASCs from the same tissue can effectively uphold their intrinsic cellular phenotype while substantially mitigating the risk of perturbations in their epigenetic memory. Thus, this method stands as a promising avenue for obtaining stem cells that closely mirror their tissue of origin, thereby ensuring the preservation of their characteristic epigenetic signatures. This is advantageous, given that induced cells inherit numerous components of epigenetic memory from donor tissues, which represents a potential safety concern in the clinic. Such precision in epigenetic maintenance holds particular significance in the realm of regenerative medicine, as it fosters the development of cells that exhibit a heightened resemblance to their in vivo counterparts, enhancing their suitability for therapeutic applications and tissue regeneration.

[0151] Although the results of SIST obtained from cells of mesodermal origin were reproduced in human endoderm and ectoderm cells, spheres formed by human hepatocytes and keratinocytes were less dense and smaller than those derived from mouse and human fibroblasts. Particularly noteworthy is the observation that the hepatocyte spheroids exhibited a less densely packed morphology with greater fragility than that of spheroids derived from fibroblasts. This could be due to differences in the basic growth requirements of somatic cells from diverse germ layer origins and could potentially be overcome via further modifications of the culture media. Furthermore, prior studies such as those cited utilized 10% fetal bovine serum as a supplement for hepatocyte spheroid generation, while this study opted for a culture medium enriched with defined growth factors, including hHGF, bFGF and hIGF-1. Despite the various limitations encountered in this study, it is important to highlight that the expression of hepatocyte stem cell markers remained discernible in the spheroids that had incurred breakage. Certainly, more work is required to improve the efficiency of ASC-enriched sphere formation from somatic cells derived from diverse tissues. Nevertheless, the results suggest that normal somatic cells can represent a rapid and high-yield source of ASCs, which could have important implications for regenerative medicine.38

[0152] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

39 CLAIMS 1. A method of generating adult stem cells from somatic cells comprising: a. treating a culture of somatic cells isolated from a donor tissue with a combination of growth factors and / or chemical compounds; and b. inducing reprogramming of the isolated somatic cells by promoting cell to cell contact, wherein cell-to-cell contact of the isolated somatic cells facilitates generation of aggregates comprising adult stem cells.

2. The method of claim 1, wherein the reprogramming of somatic cells occurs by inducing a stem-cell like state in the isolated somatic cells through cell-to-cell contact.

3. The method of claim 1, wherein the reprogramming of somatic cells is induced by subjecting the cells to a suspension culture technique which is devoid of any membrane or substate to attach on.

4. The method of claim 1 or 2, wherein the suspension culture technique increases / promotes cell-to-cell contact.

5. The method of claim 3, wherein the suspension culture technique is hanging drop culture technique, g-force culture technique, centrifugation culture technique, shaker flask culture technique, spinner flask culture technique, agitation culture technique, closed continuous culture technique, open continuous culture technique, gravity-controlled culture technique, or magnetic stirrer culture technique.

6. The method of any one of claims 1-5, wherein aggregates comprising adult stem cells display stem cell like characteristics.

7. The method of any one of claims 1-6, wherein the isolated somatic cells form aggregates of adult stem cells from which they were originally derived.

8. The method of claim 7, wherein the isolated somatic cells from mesoderm forms aggregates of mesodermal stem cells, the isolated somatic cells from ectoderm forms40 aggregates of ectodermal stem cells, and wherein isolated somatic cells from the endoderm forms aggregates of endodermal stem cells.

9. The method of claim 8, wherein the aggregates of adult stem cells display stem cell like characteristics such as self-renewal capacity and expression of tissue specific surface markers.

10. The method of claim 9, wherein the expression of surface markers is determined by immunofluorescence staining.

11. The method of any one of claims 8-10, wherein the mesodermal stem cells express surface markers such as CD73, CD105, CD106, CD146, CD166, or STRO-1; or wherein the endodermal stem cells express surface markers such as CD117, CD133, or EPCAM; or wherein the ectodermal stem cells express surface markers such as TfR / CD71 or p63 / TP73L.

12. The method of any one of claims 1-11, wherein the method further comprises: c. subjecting the aggregates of adult stem cells to target-tissue specific growth conditions to promote their differentiation into a target-tissue.

13. The method of claim 12, wherein the target-tissue specific growth conditions comprise a target-tissue specific differentiation medium with essential nutrients and growth factors.

14. The method of claim 12 or 13, wherein the differentiation of stem cells to the target-tissue is confirmed by the expression of target tissue-specific proteins.

15. The method of any of claim 12-14, wherein the aggregates of mesodermal stem cells differentiate to form a mesodermal tissue, wherein the aggregates of ectodermal stem cells differentiate to form an ectodermal tissue, and wherein the aggregates of endodermal stem cells differentiate to form an endodermal tissue.

16. The method of any one of claims 1-15, wherein the isolated stem cells aggregate to form spheroids.41 17. The method of claim 1, wherein the method comprises prior to step (a): d. isolating somatic cells from the donor tissue; e. culturing the isolated somatic cells in a suitable growth medium; f. allowing the isolated somatic cells to grow in the growth medium until they reach optimal confluency; and g. detaching the confluent somatic cells by treating with a proteolytic enzyme.

18. The method of claim 17, wherein the steps (a) and (b) immediately follow the detachment of somatic cells from the growth medium to prevent apoptosis thereby triggering the reprogramming process.

19. The method of claim any one of claims 1-18, wherein the isolated somatic cells are mesodermal somatic cells, osteoblasts, adipocytes, chondrocytes, myocytes, fibroblasts, endodermal somatic cells, hepatic stellate cells, beta cells, skeletal muscle cells, satellite cells, ectodermal somatic cells, keratinocytes, or a combination thereof.

20. The method of any one of claims 1-19, wherein the donor tissue is an ectodermal tissue (such as skin tissue), a mesodermal tissue (such as adipose tissue, muscle tissue), or an endodermal tissue (such as liver tissue, a pancreatic tissue), or a combination thereof.

21. The method of any one of claims 1-20, wherein the growth factors are human epidermal growth factor (hEGF), basic fibroblast growth factor (bFGF), human insulin-like growth factor-1 (hIGF-1), rho-associated coiled-coil inhibitor (ROCKi), human hepatocyte growth factor (hHGF), mesodermal-specific growth factors, endodermal-specific growth factors, ectodermal-specific growth factors or a mixture of any of the above.

22. The method of any one of claims 1-21, wherein the chemical compound is rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, mesodermal-specific chemical compound, endodermal-specific chemical compound, ectodermal-specific chemical compound or a mixture of any of the above.

23. The method of claim 17, wherein the isolated somatic cells are maintained in the growth medium at optimal culture conditions.42 24. The method of claim 23, wherein the optimal culture conditions comprise a pH ranging from 5.5-7.5, a temperature ranging from 35C - 40C, and a growth medium comprising 3-7% CO2, 19-23% O2, and 72-76% N2 25. The method of claim 17, wherein the growth medium is DMEM / F-12 supplemented with 10% Fetal bovine serum (FBS), Williams' Medium E supplemented with 10% Fetal bovine serum (FBS), Williams' Medium E supplemented with 10% Fetal bovine serum (FBS) and additive nutrients, keratinocyte serum free medium (k-sfm), or EpiLife™ Medium supplemented with 60 μM calcium from GIBCO and 10% Fetal bovine serum (FBS), a culture medium supplemented with MesenCult™-ACF Chondrogenic Differentiation Kit, any culture medium comprising essential nutrients for the growth of somatic cells, or a serum-free medium comprising essential nutrients for the growth of somatic cells.

26. The method of claim 17, wherein the optimal confluency ranges from 70% - 90%.

27. The method of claim 13, wherein the differentiation medium is DMEM / F-12 supplemented with 10% Fetal bovine serum (FBS), Williams' Medium E supplemented with 10% Fetal bovine serum (FBS), Williams' Medium E supplemented with 10% Fetal bovine serum (FBS) and additive nutrients, keratinocyte serum free medium (k-sfm), or EpiLife™ Medium supplemented with 60 μM calcium from GIBCO and 10% Fetal bovine serum (FBS), a culture medium supplemented with MesenCult™-ACF Chondrogenic Differentiation Kit, any culture medium that supports differentiation of endodermal, ectodermal or mesodermal cells, or a serum-free medium mesodermal-specific growth factors, endodermal-specific growth factors, ectodermal-specific growth factors or a mixture of any of the above.

28. The method of claim 1, wherein the isolated somatic cells are treated with a combination of target tissue-specific growth factors.

29. The method of any one of claims 1-28, wherein the method is for generating an ectodermal tissue (such as skin tissue, epithelial lining of mouth, tooth enamel etc.) from isolated ectodermal somatic cells, an endodermal tissue (such as epithelial lining of respiratory system, liver tissue, pancreatic tissue etc.) from isolated endodermal somatic cells or a43 mesodermal tissue (such as muscle tissue, notochord skeletal tissue, lymphatic system tissue etc.) from isolated mesodermal somatic cells.

30. The method of any one of claims 1-28, for tissue-replacement treatment, for tissue repair treatment, for stem-cell research, for genetic research, for drug testing, for disease modelling, for stem cell therapy, or for developing a stem cell bank.

31. Adult stem cells generated from isolated somatic cells using the method of claims 1-28.

32. Use of adult stem cells of claim 31, for tissue-replacement treatment, for tissue repair treatment, for stem-cell research, for genetic research, for drug testing, for disease modelling, for stem cell therapy, or for developing a stem cell bank.

33. Use of isolated somatic cells from a donor tissue for generating an ectodermal tissue (such as skin tissue, epithelial lining of mouth, tooth enamel etc.) from isolated ectodermal somatic cells, an endodermal tissue (such as epithelial lining of respiratory system, liver tissue, pancreatic tissue etc.) from isolated endodermal somatic cells or a mesodermal tissue (such as muscle tissue, notochord skeletal tissue, lymphatic system tissue etc.) from isolated mesodermal somatic cells, using the method of any one of claims 1-28.

34. A kit for generating adult stem cells or a target tissue comprising: a. a culture of confluent isolated somatic cells; b. a growth medium for supporting the growth of isolated somatic cells; c. an apparatus for suspension culture technique such as hanging drop or g-force centrifugation; and d. a differentiation medium for promoting differentiation of the isolated somatic cells to the adult stem cells or the target tissue.

35. A method of generating adult stem cells for treating stem cell disorders, for treating tissue damage, for treating genetic disorders, or for treating diseases, comprising the method of claims 1-28.