Therapeutic use of fibroblasts to stimulate regeneration and repair of atrophied spleen

Abstract

An embodiment of the present disclosure relates to the administration of a cell population to an individual for tissue regeneration. The cell population may include fibroblasts and / or immune cells, which may be modified or unmodified. The tissue for regeneration may be the spleen.

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 316,264, filed March 3, 2022, which also claims priority to U.S. Provisional Patent Application No. 63 / 344,691, filed May 23, 2022, which further claims priority to U.S. Provisional Patent Application No. 63 / 383,500, filed November 13, 2022, all of which are incorporated by reference in their entireties herein.

[0002] I.Technical field The present disclosure relates to at least the fields of medicine, immunology, cell biology, and molecular biology. [Background technology]

[0003] II. Background Fibroblasts are no longer merely structural components of organs but are now considered to be dynamic participants in immune processes: they create an environment that influences the migration, proliferation and activity of T regulatory cells, ensuring immune tolerance [1].

[0004] The spleen is one of the important secondary lymphoid organs responsible for the rapid response of the immune system to blood-borne pathogens and for maintaining long-term adaptive responses to such pathogens. The spleen is also a key organ for iron metabolism and red blood cell homeostasis. It also serves as an important reservoir for platelets and white blood cells. A variety of cell types have been identified in the spleen, including endothelial cells, mesothelial cells, reticulocytes, erythrocytes, granulocytes, monocytes, hematopoietic cells, macrophages, dendritic cells, plasma cells, CD4+ and CD8+ T cells, and migratory B cells. With aging, the structure and function of the spleen change, leading to a decreased ability to respond positively to vaccinations, increased susceptibility to viral and bacterial pathogen infections, and an increased incidence of autoimmune diseases [2]. Summary of the Invention [Problem to be solved by the invention]

[0005] As life spans increase and the proportion of elderly people in the population increases, there is a need for cell therapies that exploit the interactions between fibroblasts and other splenic cells to improve spleen productivity and extend lifespan. [Means for solving the problem]

[0006] overview The embodiments disclosed herein relate to compositions and methods for regenerating tissue in an individual. In some embodiments, one or more compositions are administered to an individual, including to regenerate tissue, stimulate proliferation of cells necessary for vital organ function, and / or reactivate production of cell products necessary to restore organ atrophy, including splenic atrophy. The administration may be useful for regenerating tissue, such as splenic tissue. In some embodiments, the regeneration includes organogenesis and / or T cell development and / or B cell development. In some embodiments, when the tissue includes the spleen, the regeneration includes reversing splenic atrophy. In some embodiments, the regeneration includes tissue differentiation, such as splenic differentiation. In certain embodiments, the regeneration includes tissue recruitment, differentiation of stem cells into tissue epithelial cells and / or tissue fibroblasts, and / or expansion of tissue epithelial cells and / or tissue fibroblasts. In certain embodiments, a combination of all or at least some of the above activities is encompassed in the regeneration of the tissue. The tissue fibroblasts may optionally include splenic reticular fibroblasts.

[0007] Certain embodiments relate to cell populations and their use as cell therapy. The cell population may include modified and / or unmodified fibroblasts (including fibroblasts; fibroblast-like cells; lysates from fibroblasts; conditioned medium from fibroblast cultures; and / or extracellular vesicles comprising exosomes, microvesicles, apoptotic bodies or other fragments or biological components of fibroblasts). The cell population may include modified immune cells and / or unmodified immune cells. The cell population may include fibroblast derivatives (including extracellular vesicles comprising exosomes, microvesicles, apoptotic bodies or other fragments or biological components of fibroblasts). In some embodiments, the cell population includes unmodified fibroblasts (including lysates from fibroblasts and / or conditioned medium from fibroblast cultures, including extracellular vesicles comprising exosomes, microvesicles, apoptotic bodies or other fragments or biological components of fibroblasts), unmodified immune cells, activated fibroblasts, activated immune cells, fibroblast derivatives, immune cell derivatives, or combinations thereof. Fibroblasts (including fibroblasts; fibroblast-like cells; lysates from fibroblasts; conditioned medium from fibroblast cultures; and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or any other fragments or biological components of fibroblasts) and / or immune cells can be activated by exposing the cells to one or more agents capable of activating the cells. Fibroblasts (including fibroblasts; fibroblast-like cells; lysates from fibroblasts; conditioned medium from fibroblast cultures; and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or any other fragments or biological components of fibroblasts) and / or immune cells can be activated by exposing the cells to one or more agents capable of activating the cells, thereby producing activated fibroblasts and / or activated immune cells, respectively. In some embodiments, the agent(s) include one or more nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, hormones, or combinations thereof. The cell population can be activated in vitro or ex vivo.

[0008] In some embodiments, fibroblasts are activated in vivo, ex vivo, or a combination of both by selecting activated fibroblasts that express and release, or express, one or more of the following factors, such as CCL21, podoplanin, CCL19, IL-7, IL-33, DES, MAdCAM-1, CXCL13, BAFF, IL-6 [3-10].

[0009] In some embodiments, fibroblasts can be activated to express transcription factors such as SPIB, WT1, STAT1, STAT2, IRF9 (ISFG3), and ETS using activation methods using RNA and / or transcription factor modulation [11, 12][13-16].

[0010] In some embodiments, the activation method includes activation of fibroblasts with RNA and / or transcription factors to ensure increased expression and secretion of Bst2, CXCL9, CXLX10, Ly6c, Sca-1, Timd4, and Fabp5 [13, 17].

[0011] In some embodiments, RNA and / or epigenetic modulation can be used to increase expression of one or a combination of two or more fibroblast-associated genes, such as Col1a1, Col3a1, Col6a1, Dnc, Lum, Mgp, Sparc, Spering1, Serpinh1, Htra1, Htra3, Mmp2, and / or Mmp3, in vivo or ex vivo.

[18]

[0012] In some embodiments, the cell population may be autologous, allogeneic, xenogeneic, syngeneic, or a combination thereof with respect to the individual.

[0013] In some embodiments, the cell population is administered systemically to an individual, including at least an individual in need of regenerating at least a portion of a spleen, and / or locally to the spleen of the individual.

[0014] Certain embodiments relate to methods, including in vitro methods, for producing activated cell populations, such as activated fibroblasts, derivatives of activated fibroblasts (including extracellular vesicles containing exosomes, microvesicles, apoptotic bodies or any other fragments or biological components of fibroblasts), activated immune cells, derivatives of activated immune cells, or combinations thereof. In some embodiments, the methods include exposing fibroblasts (including fibroblasts; fibroblast-like cells; lysates from fibroblasts; conditioned medium from fibroblast cultures; and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies or any other fragments or biological components of fibroblasts) and / or immune cells to one or more conditions that induce activation. Activation of fibroblasts, derivatives of activated fibroblasts (including extracellular vesicles containing exosomes, microvesicles, apoptotic bodies or any other fragments or biological components of fibroblasts), activated immune cells, and / or derivatives of activated immune cells can render the cell population capable of regenerating the spleen. The activated cells may be autologous, allogeneic, xenogeneic, syngeneic with respect to the individual, or the population may be comprised of a combination thereof. In some embodiments, the cells are activated by conditions including one or more nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, hormones, or combinations thereof.

[0015] In some embodiments, tissue regeneration includes stimulating, improving, or increasing the production and / or activity of immune cells in the spleen.

[0016]

[0013] Embodiments of the present disclosure include a method of regenerating tissue in an individual, the method comprising administering to the individual an effective amount of a cell population; wherein the tissue comprises the individual's spleen; the cell population comprises unmodified or modified fibroblasts (including fibroblasts; fibroblast-like cells; lysates from fibroblasts; conditioned media from fibroblast cultures; and / or extracellular vesicles comprising exosomes, microvesicles, apoptotic bodies or any other fragment or biological component of fibroblasts), unmodified or modified immune cells, activated or non-activated fibroblasts (including fibroblasts; fibroblast-like cells; lysates from fibroblasts; conditioned media from fibroblast cultures; and / or extracellular vesicles comprising exosomes, microvesicles, apoptotic bodies or any other fragment or biological component of fibroblasts), activated or non-activated immune cells, fibroblast derivatives (including extracellular vesicles comprising microvesicles, apoptotic bodies or any other fragment or biological component of fibroblasts), immune cell derivatives, or combinations thereof, thereby resulting in regeneration of splenic tissue in the individual. In some cases, the cell population is activated by exposing the cells to one or more agents capable of activating the cells prior to and / or during administration. The agents may include one or more nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, hormones, or combinations thereof. In some cases, the cell population is activated in vitro, in vivo, or ex vivo, and the cell population may be autologous, allogeneic, xenogeneic, syngeneic, or combinations thereof with respect to the individual. In any method, a step may further include administering one or more adjuvants to the individual, such as chemical adjuvants, viral adjuvants, bacterial adjuvants, or combinations thereof. In any case, the adjuvant may include encapsulated RNA, microRNA, siRNA, or combinations thereof.

[0017] In certain embodiments, regeneration comprises organogenesis and / or T cell development and / or B cell development. In certain embodiments, regeneration comprises reversing splenic atrophy. In certain embodiments, regeneration comprises reactivating or stimulating production of splenic immune cells. In certain embodiments, regeneration comprises tissue differentiation. In certain embodiments, regeneration comprises tissue recruitment, differentiation of stem cells into tissue epithelial cells and / or tissue fibroblasts, and / or expansion of tissue epithelial cells and / or tissue fibroblasts.

[0018] In certain embodiments of any of the compositions and / or methods, the tissue fibroblasts comprise splenic reticular fibroblasts.

[0019] Methods in which the activated cell population is administered to one or more individuals may include administering the population systemically to the individuals or administering the population locally to the spleen of the individuals.

[0020] In certain embodiments, there is provided an in vitro method of producing activated or non-activated fibroblasts (including fibroblasts; fibroblast-like cells; lysates from fibroblasts; conditioned medium from fibroblast cultures; and / or extracellular vesicles comprising exosomes, microvesicles, apoptotic bodies or any other fragments or biological components of fibroblasts), derivatives of activated fibroblasts (including extracellular vesicles comprising exosomes, microvesicles, apoptotic bodies or any other fragments or biological components of fibroblasts), activated or non-activated immune cells, derivatives of activated or non-activated immune cells, or combinations thereof, comprising: Methods are provided that include exposing fibroblasts (including fibroblasts; fibroblast-like cells; lysates from fibroblasts; conditioned medium from fibroblast cultures; and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies or any other fragments or biological components of fibroblasts) and / or immune cells to conditions that induce activation, wherein the activated fibroblasts, derivatives of activated fibroblasts (including extracellular vesicles containing exosomes, microvesicles, apoptotic bodies or any other fragments or biological components of fibroblasts), activated immune cells, and / or derivatives of activated immune cells include activity for regenerating spleen tissue. The activated or non-activated fibroblasts (including fibroblasts; fibroblast-like cells; lysates from fibroblasts; conditioned medium from fibroblast cultures; and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or any other fragments or biological components of fibroblasts), and / or the activated or non-activated immune cells may be autologous, allogeneic, xenogeneic, or syngeneic with respect to the spleen. In certain embodiments, regeneration includes stimulating, improving, and / or increasing the production and / or activity of immune cells in the spleen. In the method, the condition may include one or more nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, hormones, or combinations thereof.

[0021] In one embodiment, a method is provided for stimulating, improving, or increasing splenic cell production and / or activity in an individual, the method comprising providing to the individual an effective amount of activated fibroblasts, derivatives of activated fibroblasts, activated immune cells, derivatives of activated immune cells, or combinations thereof.

[0022] 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. Further, a composition of the present disclosure can be used to achieve a method of the present disclosure.

[0023] The foregoing has outlined, rather broadly, the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described below which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the concept and specific embodiments disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be appreciated by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the present invention, both as to its organization and method of operation, together with further objects and advantages, will be better understood from the following description.

[0024] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Further, a composition of the invention can be used to achieve a method of the invention.

[0025] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Detailed Description I. Definition Examples Following long-standing patent law practice, the words "a" and "an" herein, when used in conjunction with the word comprising, including in the claims, refer to "one or more." Some embodiments of the present disclosure may consist of, or consist essentially of, one or more of the elements, method steps, and / or methods of the present disclosure. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

[0027] The term "about" or "approximately" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by 30%, 25%, 20%, 25%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In certain embodiments, the term "about" or "approximately" when preceding a numerical value indicates a value within a range of plus or minus 15%, 10%, 5%, or 1%. In reference to biological systems or processes, the term may mean within an order of magnitude, preferably within 5-fold, more preferably within 2-fold, of the value. Unless otherwise specified, the term "about" means within an acceptable error range for the particular value.

[0028] As used herein, the term "activated immune cells" refers to immune cells that have been treated with one or more stimuli that can induce one or more changes within the cell, including metabolic, immunological, epigenetic, growth factor secretion, surface marker expression, and microvesicle production and excretion.

[0029] The term "administered" or "administering" as used herein refers to any method of providing a composition to an individual so that the composition produces the intended effect in the patient. For example, one method of administration is by an indirect mechanism using a medical device such as, but not limited to, a catheter, an applicator gun, a syringe, and the like. A second exemplary method of administration is by a direct mechanism such as local tissue administration, oral ingestion, a transdermal patch, topical, inhalation, suppository, and the like.

[0030] As used herein, "allogeneic" refers to tissues or cells from one or more individuals of the same species, but from another body, that are or may become immunologically incompatible in their natural environment.

[0031] As used herein, "autologous" refers to tissues or cells derived or transplanted from the body of the same individual (i.e., autologous blood donation, autologous bone marrow transplant).

[0032] As used herein, an "agent" refers to one or more small molecules, nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, and / or hormones.

[0033] As used herein, "xenogeneic" refers to tissues or cells derived from a species different from that of the patient.

[0034] "Cell culture" refers to an artificial in vitro system that contains viable cells, whether quiescent, senescent, or (actively) dividing. In cell culture, cells are grown and maintained at an appropriate temperature, typically 37°C, in an atmosphere that typically contains oxygen and CO2. However, culture conditions can vary widely from cell type to cell type, and varying conditions for a particular cell type may result in the expression of a different phenotype. The factor most often varied in culture systems is the growth medium. Growth medium varies in nutrients, concentrations of growth factors, and the presence or absence of other components. Growth factors used to supplement the medium are often derived from animal blood, such as calf serum.

[0035] Throughout this specification, unless the context dictates otherwise, the words "comprise", "comprises" and "comprising" are understood to mean the inclusion of the recited step or element or steps or elements, but not the exclusion of other steps or elements or steps or elements. "Consisting of" means including and limited to what follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are required or essential, and other elements may not be present. "Consisting essentially of" means including the elements recited after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but other elements are optional and may or may not be present depending on whether they affect the activity or action of the recited elements.

[0036] The term "individual" as used herein refers to a human or animal that may or may not be housed in a medical facility, or may be treated as an outpatient in a medical facility. An individual may receive one or more medical compositions via the Internet. An individual may include any age of human or non-human animal, and thus includes both adults and juveniles (i.e., children), and infants. As the term "individual" is not intended to imply a need for medical treatment, an individual may participate in an experiment, whether clinical or in support of basic science research, whether voluntarily or involuntarily. The terms "subject" or "individual" may be used interchangeably and refer to any organism or animal that is the subject of a method or material, including mammals, such as humans, laboratory animals (primates, rats, mice, rabbits, etc.), farm animals (cows, sheep, goats, pigs, turkeys, chickens, etc.), household pets (dogs, cats, rodents, etc.), horses, and transgenic non-human animals.

[0037] Throughout this specification, reference to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, means that the particular features, structures, or characteristics described in connection with the embodiment are included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0038] The terms "reduce," "inhibit," "diminish," "suppress," "decrease," "prevent," and grammatical equivalents (including "lower," "smaller," etc.), when referring to the onset of a symptom in an untreated subject compared to a treated subject, mean that the amount and / or magnitude of the symptom in the treated subject is lower than the amount and / or magnitude of the symptom in the untreated subject by any amount recognized as clinically appropriate by medically trained personnel. In one embodiment, the amount and / or magnitude of the symptom in the treated subject is at least 10% lower, at least 25% lower, at least 50% lower, at least 75% lower, and / or at least 90% lower than the amount and / or magnitude of the symptom in the untreated subject.

[0039] The term "unmodified" as used herein when referring to a cell or cell population may refer to cells that have been cultured only under standard conditions for that cell type, without exposure to agents that may alter the intrinsic properties of that cell type (except those that may inherently change during standard cell culture). Unmodified cells are composed only of endogenous genetic material, i.e., unmodified cells do not contain transgenes. In some embodiments, it refers to cells that have not been manipulated in any way or that have simply been cultured. In a specific embodiment, a "modified" cell is a cell that has been stimulated to the point of differentiation into another cell type, and may no longer have the morphology of the starting material. This may apply to MSCs and fibroblasts, for example.

[0040] As used herein, the term "transplantation" refers to the process of taking living tissue or cells and transplanting them into another part of the body or into another body.

[0041] "Treatment", "treat" or "treating" refers to a method of reducing the effects of a disease or condition. Treatment can also refer to a method of reducing the disease or condition itself, not just the symptoms. Treatment can be any reduction from pre-treatment levels, including but not limited to complete ablation of the disease, condition, or symptoms of the disease or condition. Thus, in the disclosed methods, "treatment" can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or disease progression, including a reduction in the severity of at least one symptom of the disease. For example, the disclosed methods for reducing the immunogenicity of a cell are considered to be therapeutic if there is a detectable reduction in the immunogenicity of the cell compared to pre-treatment levels in the same subject or in a control subject. Thus, the reduction may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount therebetween, compared to native or control levels. It is understood and contemplated herein that "treatment" does not necessarily mean curing a disease or condition, but rather means improving the prognosis of a disease or condition. In specific embodiments, treatment refers to reducing the severity or extent of at least one symptom, and may alternatively or additionally refer to delaying the onset of at least one symptom.

[0042] II. Fibroblasts Embodiments of the present disclosure include compositions, methods, and / or systems for introducing fibroblasts (including fibroblasts, fibroblast-like cells, lysates from fibroblasts, conditioned media from fibroblast cultures, and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or any other fragments or biological components of fibroblasts) into the spleen and / or modifying existing fibroblasts (including fibroblasts, fibroblast-like cells, lysates from fibroblasts, conditioned media from fibroblast cultures, and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or any other fragments or biological components of fibroblasts) in the spleen and regenerating their function [3, 4]. In certain embodiments, the present disclosure relates to the interaction of a first type of cells with a second type of cells and / or certain agents. The interaction may include one or more modifications to the first and / or second type of cells as a result of the interaction. Embodiments of the present disclosure include compositions, methods, and systems in which fibroblasts (including fibroblasts, fibroblast-like cells, lysates from fibroblasts, conditioned media from fibroblast cultures, and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or any other fragments or biological components of fibroblasts) are modified by exposure to one or a combination of specific cells and / or specific agents including nucleic acids, cytokines, chemokines, or growth factors. In some embodiments, introduction of these modified or unmodified fibroblasts (including fibroblasts, fibroblast-like cells, lysates from fibroblasts, conditioned media from fibroblast cultures, and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or any other fragments or biological components of fibroblasts) into the spleen regenerates necessary functional cells such as splenic reticular fibroblasts, splenic fibroblasts, splenic macrophages, stromal organizer cells, and / or other non-hematopoietic cells. In some embodiments, introduction initiates a foundation for new tissue growth [5].The regeneration of these cells occurs through several possible steps, including: differentiation of modified or unmodified administered fibroblasts (including fibroblasts, fibroblast-like cells, lysates from fibroblasts, conditioned medium from fibroblast cultures, and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies or any other fragments or biological components of fibroblasts) into reticular fibroblasts, splenic fibroblasts, stromal organizer cells, and / or discontinuous splenic epithelium; activation of senescent splenic reticular epithelium, fibroblasts, and / or stromal organizer cells (including cell-cell contact with modified or unmodified fibroblasts); differentiation of modified or unmodified fibroblasts into splenic microenvironment; Activation of senescent splenic reticular epithelium, fibroblasts, and / or stromal organizer cells by products excreted from cells; differentiation of local stem cells into splenic reticular fibroblasts, splenic fibroblasts, splenic macrophages, and / or stromal organizer cells by the direct / indirect action of infused modified / unmodified fibroblasts and / or via products excreted from modified / unmodified fibroblasts; and / or proliferation of the small number of splenic reticular fibroblasts, splenic fibroblasts, splenic macrophages, and stromal organizer cells remaining in spleens affected by injury or age-related atrophy to sufficient numbers, which may affect and significantly improve splenic function.

[0043] In some embodiments, reversal of splenic atrophy is monitored by magnetic resonance imaging (MRI), such as determining the fat-free fraction of the spleen.In addition, complete blood count assessment, fluorescence-activated cell sorting (FACS) sorting characterization of blood cells, and / or flow cytometry can provide the necessary metrics to determine changes in the spleen and / or monitor the effects of modified and / or unmodified fibroblasts injected into the atrophied spleen.

[0044] Certain embodiments relate to compositions, methods, and / or systems in which exposure to fibroblasts and / or specific agents excreted from fibroblasts modifies specific cells, including any of the spleen cells disclosed herein. In certain embodiments, interaction of fibroblasts with one or more other types of cells (and optionally, the interaction also includes one or more specific agents) results in modification of the fibroblasts and / or one or more cells of the spleen. [6] In specific embodiments, the cells of the spleen include at least immune cells.

[0045] In certain embodiments, introduction of fibroblasts (including fibroblasts, fibroblast-like cells, lysates from fibroblasts, conditioned medium from fibroblast cultures, and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or other fragments or biological components of fibroblasts) into the spleen results in modification of fibroblasts and / or other cells found in the spleen to stimulate regeneration of splenic structure and function. [7] In certain aspects, exposure of fibroblasts (including fibroblasts, fibroblast-like cells, lysates from fibroblasts, conditioned medium from fibroblast cultures, and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or other fragments or biological components of fibroblasts) and / or agents produced therefrom results in modification of one or more immune cells and / or agents produced therefrom results in modification of fibroblasts. In specific embodiments, the exposure also includes one or more agents, which may or may not be provided exogenously, as in other cases, are endogenous to the environment and / or cells and / or tissues.

[0046] In certain embodiments, the disclosed method occurs in vitro, such as in culture, or ex vivo. In certain embodiments, the disclosed method occurs by the hand of man and does not encompass normal or random occurrence in the body. The disclosed method is, in certain embodiments, non-natural. In certain embodiments, the concentration of cells used in the method of exposing one type of cell to another type of cell does not occur in nature, does not occur randomly in nature. In specific embodiments, the concentration of one or more agents used in the method of exposing one or more agents to one or more types of cells does not occur in nature, does not occur randomly in nature. Any type of cell modification encompassed by the disclosure that occurs ex vivo or in vitro does not occur in the same way in nature in vivo. In certain embodiments, tissue biopsies from donors are used to isolate, characterize, and optionally activate, expand, and reintroduce one or a combination of cells into the donor for the purpose of reactivating an atrophied spleen [8].

[0047] The present disclosure encompasses therapeutic uses of cells including epithelial cells, thymocytes, dendritic cells, macrophages, B lymphocytes, myoid cells, endothelial cells, fibroblasts, immune cells, splenic reticular fibroblasts, splenic fibroblasts, splenic macrophages, stromal organizer cells, and mixtures thereof. In at least some cases, the fibroblasts are modified, such as chemically, physically, and / or epigenetically activated, prior to exposure to one or more immune cells and other cells found in the spleen, or exposed to conditions not normally found in the body. In certain embodiments, the immune cells or derivatives thereof are modified, such as activated, prior to exposure to the fibroblasts.

[0048] The embodiments of the present disclosure provide a means to utilize fibroblasts (including fibroblasts, fibroblast-like cells, and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or any other fragments or biological components of fibroblasts) as autologous, allogeneic, xenogeneic, or syngeneic gene therapy cells through modification of culture conditions. In one embodiment of the present disclosure, the fibroblasts are extracted from a low immunogenic source (e.g., placental fibroblasts, fibroblasts derived from fetal membrane tissue, fibroblasts derived from umbilical cord blood, etc.).

[0049] The present disclosure provides a method for reducing the immunogenicity of certain types of fibroblasts.Fibroblasts can be derived from various tissues or organs, including but not limited to skin, heart, blood vessels, bone marrow, skeletal muscle, liver, pancreas, brain, foreskin, and can be obtained during biopsy (if appropriate) or autopsy.In some aspects, the cells include fibroblasts and can be of fetal, neonatal, adult, or combinations thereof.

[0050] Fibroblasts (including fibroblasts, fibroblast-like cells, lysates from fibroblasts, conditioned media from fibroblast cultures, and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or any other fragments or biological components of fibroblasts) for use in any of the methods of the present disclosure may, in certain embodiments, be exposed to a particular media component(s).

[0051] Certain embodiments relate to methods of producing activated or non-activated fibroblasts (including fibroblasts, fibroblast-like cells, lysates from fibroblasts, conditioned medium from fibroblast cultures, and / or extracellular vesicles comprising exosomes, microvesicles, apoptotic bodies, or any other fragments or biological components of fibroblasts), or activated or non-activated immune cells, or activated or non-activated immune cell or fibroblast derivatives, for introduction into the spleen. Introduction into the spleen may stimulate, improve, and / or increase the production or activity of immune cells, iron metabolism, and / or red blood cell homeostasis. In some embodiments, the method is performed in vivo. In some embodiments, the method is performed in vitro. In some embodiments, the method is performed ex vivo.

[0052] In some embodiments, the fibroblasts are autologous. In some embodiments, the fibroblasts are allogeneic. In some embodiments, the fibroblasts are xenogeneic.

[0053] Certain embodiments relate to the introduction of fibroblasts (including fibroblasts, fibroblast-like cells, lysates from fibroblasts, conditioned medium from fibroblast cultures, and / or extracellular vesicles containing exosomes, microvesicles, apoptotic bodies, or other fragments or biological components of fibroblasts), drug-activated fibroblasts, and / or fibroblasts and one or more immune cells and / or derivative drugs into the spleen. The spleen may be atrophic. The introduction may induce organogenesis, T cell development, and / or reversal of splenic atrophy. The introduction may induce differentiation of the tissue to reverse splenic atrophy.

[14] The introduction may induce cell recruitment and differentiation of stem cells to the red pulp, white pulp, or splenic marginal zone.

[12] This introduction can induce proliferation of cells already present in the spleen, including splenic endothelial cells, mesothelial cells, reticular cells, reticular fibroblasts, splenic fibroblasts, splenic macrophages, and interstitial organizer cells [15-17].

[0054] Certain embodiments relate to the use of one or more adjuvants in combination with fibroblasts to activate the immune system. The adjuvants may be chemical, viral, or bacterial product-based.

[0055] Certain embodiments relate to the combination of encapsulated RNA, microRNA, or other RNAi molecules or systems with fibroblasts, which may locally stimulate regeneration of splenic tissue or reverse splenic atrophy.

[0056] III. Drugs Certain embodiments relate to specific agents and methods of using such agents. The agents may include any agent useful in the methods and compositions encompassed herein. The cell populations of the present disclosure may be exposed to one or more agents for one or a variety of purposes. The agents may, for example, activate the cells of the cell population. In some embodiments, cell populations including unmodified cell populations and / or cell populations activated by an agent and / or derivatives of the cell populations are administered to an individual with an agent. The administration may induce differentiation of the tissue to reverse tissue involution. The administration may induce tissue recruitment and differentiation of stem cells into epithelial cells of the tissue and / or fibroblasts of the tissue, such as splenic reticular fibroblasts. The administration may induce proliferation of epithelial cells and / or fibroblasts present in the tissue.

[0057] In some embodiments, the agent comprises an adjuvant. In some embodiments, the adjuvant is administered to an individual to activate the immune system of the individual. The adjuvant can be used in combination with the cell population to activate the immune system. The adjuvant can be chemical, viral, bacterial product-based, or a combination thereof.

[0058] In certain embodiments, fibroblasts are cultured in vitro to maintain the viability and proliferation capacity of fibroblasts. The present disclosure provides modifications of known culture techniques to reduce the recognition of fibroblasts by the recipient immune system. In one embodiment, fibroblasts are cultured under xeno-free conditions, such as xeno-free medium. In some cases, the medium does not contain, for example, fetal calf serum. In specific embodiments, the present disclosure encompasses replacing fetal calf serum with one or more other agents that promote reduced immunogenicity of fibroblasts, such as human platelet-rich plasma, platelet lysate, umbilical cord blood serum, autologous serum, and / or one or more defined cytokines, such as one or a combination of fibroblast growth factor, epidermal growth factor, leukemia inhibitory factor, insulin-like growth factor, angiopoietin, and vascular endothelial growth factor.

[0059] A. Nucleic acid In certain embodiments, the nucleic acid sequences may be present in various embodiments, such as isolated segments of integrated sequences or recombinant polynucleotides and recombinant vectors, sufficient polynucleotides for use as hybridization probes, PCR or sequencing primers for identifying, analyzing, mutating or amplifying polynucleotides encoding polypeptides, antisense nucleic acids for inhibiting expression of polynucleotides, and complementary sequences of the foregoing described herein. Nucleic acids encoding epitopes are also provided. Nucleic acids encoding proteins, including fusion proteins, are also provided. Nucleic acids may be single-stranded or double-stranded and may include RNA and / or DNA nucleotides and artificial variants thereof (e.g., peptide nucleic acids).

[0060] In certain embodiments, there are polynucleotide variants that have substantial identity to the sequences disclosed herein; including at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more sequence identity, including all values ​​and ranges therebetween, compared to the polynucleotide sequences provided herein using the methods described herein (e.g., BLAST analysis using standard parameters). In certain aspects, the isolated polynucleotide will comprise a nucleotide sequence that encodes a polypeptide having at least 90%, preferably 95% or more identity over the entire length of the sequence, to the amino acid sequences described herein; or a nucleotide sequence complementary to said isolated polynucleotide.

[0061] Regardless of the length of the coding sequence itself, nucleic acid segments can be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc., and in such cases the overall length can vary considerably. Nucleic acids can be of any length. They can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 or more nucleotides in length, and / or can include one or more additional sequences, such as regulatory sequences, and / or can be part of a larger nucleic acid, such as a vector. Thus, it is contemplated that nucleic acid fragments of almost any length can be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, the nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow purification, transport, secretion, post-translational modification of the polypeptide, or to allow therapeutic benefits such as targeting or efficacy. As noted above, tags or other heterologous polypeptides may be added to the modified polypeptide coding sequence, where "heterologous" refers to a polypeptide that is not identical to the modified polypeptide.

[0062] B. Hybridization A nucleic acid that hybridizes to another nucleic acid under a specific hybridization condition. Methods for hybridizing nucleic acids are well known in the art. For example, see Current Protocols in Molecular Biology, John Wiley and Sons, NY (1989), 6.3.1-6.3.6. As defined herein, moderately stringent hybridization conditions use a pre-wash solution that includes 5x sodium chloride / sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), about 50% formamide, 6x SSC, hybridization buffer at 55°C (or other similar hybridization solutions, such as about 50% formamide and a hybridization temperature of 42°C), and washing conditions of 0.5x SSC, 0.1% SDS at 60°C. Stringent hybridization conditions include hybridization at 6×SSC at 45° C., followed by one or more washes at 0.1×SSC, 0.2% SDS at 68° C. Moreover, one of skill in the art can manipulate the hybridization and / or wash conditions to increase or decrease the stringency of hybridization, such that nucleic acids that contain nucleotide sequences that are at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to each other will typically remain hybridized to each other.

[0063] Parameters influencing the selection of hybridization conditions and guidelines for devising appropriate conditions are described, for example, by Sambrook, Fritsch, and Maniatis (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11 (1989); Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4 (1995), both of which are incorporated by reference in their entireties for all purposes) and can be readily determined by one of skill in the art, based on, for example, the length and / or base composition of the DNA.

[0064] C. Mutation Alterations can be introduced by mutation into a nucleic acid, including an endogenous or exogenous nucleic acid to any cell herein, thereby resulting in a change in the amino acid sequence of a polypeptide (e.g., an antibody or antibody derivative) that it encodes. Mutations can be introduced using any technique known in the art. In one embodiment, one or more specific amino acid residues are altered, for example, using a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues are altered, for example, using a random mutagenesis protocol. However produced, the mutant polypeptides can be expressed and screened for desired properties.

[0065] Mutations can be introduced into a nucleic acid without significantly altering the biological activity of the polypeptide it encodes. For example, nucleotide substitutions can be made that lead to amino acid substitutions of non-essential amino acid residues. Alternatively, one or more mutations can be introduced into a nucleic acid that selectively alter the biological activity of the polypeptide it encodes. See, for example, Romain Studer et al., Biochem. J. 449:581-594 (2013). For example, a mutation can quantitatively or qualitatively alter a biological activity. Examples of quantitative changes include increasing, decreasing, or eliminating an activity. Examples of qualitative changes include changing the antigen specificity of an antibody.

[0066] D. Probe In another aspect, the nucleic acid molecule is suitable for use as a primer or hybridization probe for the detection of a nucleic acid sequence. The nucleic acid molecule can contain only a portion of a nucleic acid sequence encoding a full-length polypeptide, e.g., a fragment that can be used as a probe or primer, or a fragment that encodes an active portion of a given polypeptide.

[0067] In another embodiment, the nucleic acid molecule can be used as a probe or PCR primer for a specific antibody sequence. For example, the nucleic acid molecule probe can be used in diagnostic methods, or the nucleic acid molecule PCR primer can be used to amplify a region of DNA that can be used to isolate a nucleic acid sequence for use in producing a variable domain of an antibody. See, for example, Gaily Kivi et al., BMC Biotechnol. 16:2 (2016). In a preferred embodiment, the nucleic acid molecule is an oligonucleotide. In a more preferred embodiment, the oligonucleotide is from the hypervariable regions of the heavy and light chains of the antibody of interest. In a further preferred embodiment, the oligonucleotide encodes all or part of one or more CDRs.

[0068] Probes based on the desired sequence of the nucleic acid can be used to detect the nucleic acid or similar nucleic acids, such as transcripts encoding the polypeptide of interest. The probes can contain a label group, such as a radioisotope, a fluorescent compound, an enzyme, or an enzyme coenzyme. Such probes can be used to identify cells expressing the polypeptide.

[0069] E. Inhibitory Oligonucleotides In some aspects, the present disclosure relates to inhibitory oligonucleotides that inhibit the expression of a gene of interest. The inhibitory oligonucleotides can inhibit the RNA and / or protein expression of the gene of interest. Examples of inhibitory oligonucleotides include, but are not limited to, small interfering RNA (siRNA), short hairpin RNA (shRNA), double-stranded RNA, antisense oligonucleotides, ribozymes, and oligonucleotides encoding any of the above. The inhibitory oligonucleotides can inhibit the transcription of a gene or inhibit the translation of a gene transcript in a cell. The inhibitory oligonucleotides can be 16-1000 nucleotides in length, and in certain embodiments, are 18-100 nucleotides in length. The oligonucleotide may have at least or at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 50, 60, 70, 80, or 90 nucleotides (or any range derivable therein). The oligonucleotide may be DNA, RNA, or cDNA encoding the inhibitory RNA.

[0070] Inhibitory oligonucleotides are well known in the art.For example, siRNA and double-stranded RNA are described in U.S. Patent No. 6,506,559 and U.S. Patent No. 6,573,099, and U.S. Patent Publication No. 2003 / 0051263, No. 2003 / 0055020, No. 2004 / 0265839, No. 2002 / 0168707, No. 2003 / 0159161 and No. 2004 / 0064842, all of which are incorporated herein by reference in their entirety.

[0071] Specifically, the inhibitory oligonucleotides can reduce expression of a gene of interest by at least 10%, 20%, 30%, or 40%, more specifically at least 50%, 60%, or 70%, and most specifically at least 75%, 80%, 90%, 95%, 99%, or 100% or more, or any range or value in between.

[0072] In further embodiments, there are synthetic oligonucleotides that inhibit a protein of interest. The inhibitors may be 17-25 nucleotides in length and include a 5' to 3' sequence that is at least 90% complementary to the 5' to 3' sequence of a mature mRNA. In certain embodiments, the inhibitor molecules are 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, or any range derivable therein. Additionally, the inhibitor molecules have a sequence (5' to 3') that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, or 100% complementary to the 5' to 3' sequence of a mature mRNA, particularly a mature naturally occurring mRNA, or any range derivable therein. A person skilled in the art could use as the sequence of the mRNA inhibitor a portion of the probe sequence that is complementary to the sequence of the mature mRNA, and furthermore, that portion of the probe sequence could be modified so that it is still 90% complementary to the sequence of the mature mRNA.

[0073] In some embodiments, inhibitory oligonucleotides are analogs and may contain modifications, particularly modifications that increase nuclease resistance, improve binding affinity, and / or improve binding specificity. For example, if the sugar moiety of a nucleoside or nucleotide is replaced with a carbocyclic moiety, it is no longer a sugar. Furthermore, if other substitutions are made, such as replacement of the intersugar phosphodiester bond, the resulting material is no longer a true species. All such compounds are considered to be analogs. Throughout this specification, reference to the sugar moiety of a nucleic acid species shall be understood to refer to either the true sugar or the species that occupies the structural position of the sugar of a wild-type nucleic acid. Furthermore, reference to an intersugar bond shall include the moiety that serves to link the sugar or sugar analog moiety in the manner of a wild-type nucleic acid.

[0074] The present disclosure relates to modified oligonucleotides, i.e. oligonucleotide analogs or oligonucleosides, and methods for producing modifications.These modified oligonucleotides and oligonucleotide analogs may show increased chemical and / or enzymatic stability compared to their naturally occurring counterparts.Extracellular and intracellular nucleases generally do not recognize and therefore do not bind to backbone-modified compounds.When present as protonated acid form, the absence of a negatively charged backbone may facilitate penetration into cells.

[0075] The modified internucleoside linkages replace the naturally occurring phosphodiester-5'-methylene linkage with a four-atom linking group that is intended to confer nuclease resistance and enhanced cellular uptake to the resulting compounds.

[0076] The modifications can be accomplished using a solid support, which can be manually manipulated or used in conjunction with a DNA synthesizer using methodologies commonly known to those skilled in the art of DNA synthesizers. In general, the procedure involves functionalizing the sugar moieties of two nucleosides that are adjacent to each other in a selected sequence. In the 5' to 3' sense, an "upstream" synthon, such as Structure H, is modified at its terminal 3' site, and a "downstream" synthon, such as Structure H1, is modified at its terminal 5' site.

[0077] Oligonucleosides linked by hydrazine, hydroxylamine, and other linking groups can be protected at the 5'-hydroxyl with a dimethoxytrityl group and activated at the 3'-hydroxyl for coupling with a cyanoethyldiisopropyl-phosphite moiety. These compounds can be inserted into any sequence by standard solid-phase automated DNA synthesis techniques. One of the most popular processes is the phosphoramidite technique. Oligonucleotides containing uniform backbone linkages can be synthesized using CPG solid-phase supports and standard nucleic acid synthesizers such as Applied Biosystems Inc. 380B and 394, Milligen / Biosearch 7500 and 8800. The first nucleotide (number 1 at the 3' end) is attached to a solid support such as controlled pore glass. Each new nucleotide is attached in a sequence-specific order, either manually or by an automated synthesis system.

[0078] The free amino groups can be alkylated, for example, with acetone and sodium cyanoborohydride in acetic acid. The alkylation process can be used to introduce other useful functional molecules onto the polymer. Such useful functional molecules include, but are not limited to, reporter molecules, RNA cleaving groups, groups for improving the pharmacokinetic properties of oligonucleotides, groups for improving the pharmacodynamic properties of oligonucleotides, and the like. Such molecules can be attached or conjugated to the polymer via a bond to a nitrogen atom of the backbone linkage. Alternatively, such molecules can be attached to a pendant group extending from a hydroxyl group of the sugar moiety of one or more nucleotides. Examples of such other useful functional groups are provided by WO1993007883, which is incorporated herein by reference, and the other patent applications mentioned above.

[0079] Solid supports may include those known in the art for polynucleotide synthesis, including controlled pore glass (CPG), oxalyl controlled pore glass, TentaGel Support, an aminopolyethylene glycol derivatized support, and Poros, a copolymer of polystyrene / divinylbenzene. Attachment and cleavage of nucleotides and oligonucleotides may be performed by standard procedures. As used herein, the term solid support further includes any linker (e.g., long chain alkylamines and succinyl residues) used to attach the growing oligonucleoside to a stationary phase, such as CPG. In some embodiments, the oligonucleotide may be further defined as having one or more locked nucleotides, ethylene bridged nucleotides, peptide nucleic acids, or 5'(E)-vinylphosphonate (VP) modifications. In some embodiments, the oligonucleotide has one or more phosphorothioated DNA or RNA bases.

[0080] IV. Cell therapy Certain embodiments herein include substantially homogeneous, allogeneic, or xenogeneic cell populations useful for the indications disclosed herein. The cell populations may include any of the cells disclosed herein, including modified and / or unmodified fibroblasts, modified and / or unmodified immune cells, or any modified and / or unmodified cells capable of regenerating a tissue such as the spleen. Certain embodiments herein include methods of administering the cell populations to an individual as a cell therapy.

[0081] In some embodiments, fibroblasts are engineered or stimulated to produce one or more factors and / or agents. In some embodiments, fibroblasts are engineered or stimulated to produce one or more factors and / or agents, such as leukemia inhibitory factor (LIF), brain-derived neurotrophic factor (BDNF), epidermal growth factor receptor (EGF), basic fibroblast growth factor (bFGF), FGF-6, glial-derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (GCSF), hepatocyte growth factor (HGF), IFN-γ, insulin-like growth factor binding protein (IGFBP-2), IGFBP-6, IL-1ra, IL-6, IL-8, monocyte chemotactic protein (MCP-1), mononuclear phagocyte colony-stimulating factor (M ), or a combination of these factors and / or agents. -CSF), neurotrophic factor (NT3), tissue inhibitor of metalloproteinases (TIMP-1), TIMP-2, tumor necrosis factor (TNF-β), vascular endothelial growth factor (VEGF), VEGF-D, urokinase plasminogen activator receptor (uPAR), bone morphogenetic protein 4 (BMP4), IL1-a, IL-3, leptin, stem cell factor (SCF), stromal cell-derived factor-1 (SDF-1), platelet-derived growth factor-BB (PDGFBB), transforming growth factor β (TGFβ-1) and / or TGFβ-3. Factors from the engineered or stimulated fibroblasts are present in the conditioned medium and can be collected for therapeutic use.

[0082] A.Cell culture Certain embodiments relate to the culture of any suitable cells for incorporation into the compositions and / or methods described herein for use. In some embodiments, the cells are grown and maintained at an appropriate temperature, typically 37° C., typically in an atmosphere containing oxygen and CO2. Culture conditions may vary widely from cell type to cell type, and varying conditions for a particular cell type may result in the expression of different phenotypes. The factor most often varied in culture systems is the growth medium. Growth medium varies in nutrients, growth factor concentrations, and the presence or absence of other components. Growth factors used to supplement the medium are often derived from animal blood, such as calf serum.

[0083] In some embodiments, the cells may be cultured for at least about 10 days to about 40 days, at least about 15 days to about 35 days, at least about 15 days to about 21 days, such as at least about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, or about 21 days. In some embodiments, the cells of the present disclosure may be cultured for 60 days or less, or 50 days or less, or 45 days or less. The cells may be cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days. The cells may be cultured in the presence of a liquid medium. Typically, the medium consists of a basal medium formulation known in the art. Many basal medium formulations can be used for culturing cells herein, including, but not limited to, Eagle's Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), alpha modified minimum essential medium (α-MEM), Basal Essential Medium (BME), Iscove's Modified Dulbecco's Medium (IMDM), BGJb medium, F-12 Nutrient Mixture (Ham), Liebovitz L-15, DMEM / F-12, Modified Eagle's Essential Medium (EMEM), RPMI-1640, and modifications and / or combinations thereof. The composition of the above basal medium is generally known in the art, and it is within the skill of the artisan to modify or adjust the concentration of the medium and / or medium supplements as needed for the cells being cultured. In some embodiments, the medium formulation can be Explant Medium (CEM) consisting of IMDM supplemented with 10% fetal bovine serum (FBS), 100 U / ml penicillin G, 100 μg / ml streptomycin, and 2 mmol / L L-glutamine. In other embodiments, additional basal medium formulations, such as those selected from those described above, may be employed.

[0084] Any medium capable of supporting cells in vitro can be used for culturing the cells. Media formulations capable of supporting cell growth include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), alpha modified minimum essential medium (αMEM), Roswell Park Memorial Institute Medium 1640 (RPMI Medium 1640), and the like. Typically, up to 20% fetal bovine serum (FBS) or 1-20% horse serum is added to the above media to support cell growth. However, defined media can also be used, provided that the growth factors, cytokines, and hormones required for the culture of cells are provided in the media at appropriate concentrations. Media useful in the methods of the present disclosure may contain one or more compounds of interest, including, but not limited to, antibiotics, mitogenic compounds, or differentiation compounds useful for the culture of cells. Cells can be grown at temperatures between 27°C and 40°C, for example, between 31°C and 37°C, and can be grown in a humidified incubator. Carbon dioxide concentration can be maintained at 2% to 10%, and oxygen concentration can be maintained at 1% to 22%. However, the present disclosure should in no way be construed as limited to any one method of isolating and culturing cells, rather, any method of isolating and culturing cells should be construed as being encompassed by the present disclosure.

[0085] For use in cell culture, media may be supplemented with one or more further components. For example, additional supplements may be used to provide cells with trace elements and substances necessary for optimal growth and expansion. Such supplements include insulin, transferrin, selenium salts, and combinations thereof. These components may be included in salt solutions such as, but not limited to, Hanks' Balanced Salt Solution (HBSS), Earle's Salt Solution, etc. Additionally, antioxidant supplements, e.g., β-mercaptoethanol, may be added. Many media already contain amino acids, but some amino acids may be supplemented later, e.g., L-glutamine, which is known to be less stable in solution. The medium may include, but is not limited to, antibiotic and / or antifungal compounds, typically a mixture of penicillin and streptomycin, and / or other compounds such as amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampicin, spectinomycin, tetracycline, tylosin, and zeocin. It is also contemplated to supplement the cell culture medium with mammalian plasma or serum, which often contain cellular factors and components necessary for viability and growth. The use of suitable serum substitutes is also contemplated.

[0086] Reference to a particular buffer, medium, reagent, cell, culture condition, etc., or subclass thereof, is not intended to be limiting, and should be read to include all such related material that a person skilled in the art would recognize as interesting or valuable in the particular context in which the discussion is presented. For example, it is often possible to replace one buffer system or culture medium with another, using a different but known method to achieve the same purpose for which the suggested method, material or composition is indicated for use. In certain embodiments, the cells are cultured in a cell culture system, preferably a culture vessel, containing a cell culture medium, in particular in a cell culture medium supplemented with a suitable and determined substance to protect the cells from in vitro senescence and / or to induce non-specific or specific reprogramming.

[0087] B. Cell generation Certain methods of the present disclosure relate to the culture of cells obtained from human tissue samples. In certain embodiments of the present disclosure, cells are plated on a substrate that allows cell adhesion. This can be performed, for example, by plating cells on a culture plate that presents one or more substrate surfaces that are compatible with cell adhesion. When one or more substrate surfaces are contacted with a suspension of cells (e.g., suspension in medium) introduced into the culture system, cell adhesion can occur between the cells and the substrate surface. Thus, in certain embodiments, cells are introduced into a culture system that generally features at least one substrate surface that is compatible with cell adhesion, so that plated cells can contact said substrate surface, and such embodiments include plating on a substrate that allows cell adhesion.

[0088] The cells of the present disclosure can be identified and characterized by the expression of specific marker proteins, such as cell surface markers. Detection and isolation of these cells can be achieved, for example, by flow cytometry, ELISA, and / or magnetic beads. Single-cell RNA-Seq and / or reverse transcription polymerase chain reaction (RT-PCR) can be used to quantify cell-specific genes and / or monitor changes in gene expression in response to differentiation. In certain embodiments, the marker proteins used to identify and characterize the cells are part of a list of cluster of differentiation (CD) markers previously identified in publications for various cell types and subtypes, including immune system cells. These can be selected from the group consisting of c-Kit (CD117), Nanog, Sox2, Hey1, SMA, Vimentin, Cyclin D2, Snail, E-cadherin, Nkx2.5, GATA4, CD105, CD90, CD29, CD73, Wt1, CD34, CD45, CD4, CD8, CD3, TCR, CD2, CD28, CD5, CD7, CD40L, CD19, CD20, ldb, lga, sig, CD40, MHC II, CD22, CR1 / CD35, CR2 / CD21, B7 / CD80, CD5, FcgR II / CD32, and combinations thereof.

[0089] C. Polypeptide Expression In some embodiments, nucleic acid molecules are present that code for the polypeptides or peptides of the present disclosure, and any cell encompassed herein can be modified to produce them. These can be produced by methods known in the art, such as isolated from B cells of immunized and isolated mice, phage display, expressed in any suitable recombinant expression system, or recombinantly. The nucleic acid molecules can be used to express large amounts of the polypeptide.

[0090] D. Vector In some embodiments, expression vectors containing nucleic acid molecules that encode a polypeptide of a desired sequence or a portion thereof are contemplated. In some embodiments, expression vectors containing nucleic acid molecules may encode fusion proteins, modified antibodies, antibody fragments, and probes thereof. In addition to control sequences that regulate transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well.

[0091] To express a polypeptide or peptide of the present disclosure, DNA encoding the polypeptide or peptide can be inserted into an expression vector such that the gene region is operably linked to transcriptional and translational control sequences. Typically, expression vectors used in any host cell contain sequences for plasmid or viral maintenance and for cloning and expressing foreign nucleotide sequences. Such sequences, collectively referred to as "flanking sequences", typically contain one or more of the following operably linked nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence including donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element. Such sequences and their uses are well known in the art.

[0092] E. Expression Systems Numerous expression systems exist that include at least some or all of the expression vectors described above. Prokaryotic and / or eukaryotic based systems can be adopted for use with the embodiments to produce the nucleic acid sequences, or their cognate polypeptides, proteins, and peptides. Commercially widely available systems include, but are not limited to, bacterial, mammalian, yeast, and insect cell systems. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins. An appropriate cell line or host system can be selected to ensure correct modification and processing of the expressed foreign protein. Those skilled in the art can use an appropriate expression system to express the vectors that produce the nucleic acid sequences or their cognate polypeptides, proteins, or peptides.

[0093] F. Methods of Gene Transfer Suitable methods for nucleic acid delivery to effect expression of the compositions are expected to include virtually any method by which a nucleic acid (e.g., DNA, including viral and non-viral vectors; CRISPR-Cas9) can be introduced into any cell, any tissue, or any organism, as described herein or as would be known to one of skill in the art. Such methods include direct delivery of DNA, such as by injection (U.S. Patents 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859, each of which is incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Patent No. 5,789,215, each of which is incorporated herein by reference); by electroporation (U.S. Patent No. 5,384,253, each of which is incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); using DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al., 1987); by liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); particle bombardment (PCT Application Nos. WO 94 / 09699 and WO 95 / 06128; U.S. Patent Nos. 5,610,042; 5,322,783; 5,563,055; 5,550,318; 5,538,877; and 5,538,880, each of which is incorporated herein by reference); silicon carbide fiber agitation (Kaeppler et al., 1990; U.S. Patent Nos. 5,302,523 and 5,464,765, each of which is incorporated herein by reference); Agrobacterium-mediated transformation (U.S. Patent Nos. 5,591,616 and 5,563,055, each of which is incorporated herein by reference); or PEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Patent Nos. 4,684,611 and 4,952,500, each of which is incorporated herein by reference); desiccation / inhibition-mediated DNA uptake (Potrykus et al., 1985). Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction.

[0094] In some embodiments, any cell encompassed herein can be modified to have one or more endogenous gene knockdowns or knockouts. Those skilled in the art are familiar with standard procedures for doing so, including, for example, any type of RNA interference and CRISPR technology.

[0095] G.CRISPR-mediated gene activation Various genes, including tumor necrosis factor receptor (TNFR) family, activating NF-κB receptor (RANK), CD40, lymphocyte β receptor (LTβR), fibroblast growth factor (FGF) Foxn1, and Wnt, are involved in the maturation and development of thymic epithelial cells (TECs) (Sun, Luo et al. 2013, Sun, Sun et al. 2015). TEC maturation also involves interactions and dependencies with other cells, such as thymocytes, fibroblasts, and mesenchymal stem cells, in that they can provide factors required for epigenetic activation of genes required for maturation. In this embodiment, fibroblasts and / or cells from the spleen can be epigenetically activated through CRISPR-mediated methylation of genes and promoter regions of one or a combination of genes responsible for predestined TEC activation to both the cortex and medulla of the thymus (Vojta, Dobrinic et al. 2016, Tyurin-Kuzmin, Karagyaur et al. 2018, Kang, Park et al. 2019, Nakamura, Gao et al. 2021, Singina, Sergiev et al. 2021). The same approach can be used for acetylation of genes and promoter regions to reactivate expression of genes required for TEC maturation.

[0096] H. host cell In another aspect, the use of any host cell into which a recombinant expression vector has been introduced is contemplated. An expression construct encoding a protein of interest can be transfected into a cell according to various methods known in the art. Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. Some vectors can use control sequences that allow them to be replicated and / or expressed in both prokaryotic and eukaryotic cells. In certain embodiments, the expression construct can be under the control of a promoter linked to cellular activation, such as those controlled by NFAT-1 or NF-κB. Conditions for incubating host cells to maintain the host cells and allow replication of the vector will be understood by those of skill in the art. Techniques and conditions that allow large-scale production of vectors, production of nucleic acids encoded by the vectors and their cognate polypeptides, proteins, or peptides are also understood and known.

[0097] When mammalian cells are stably transfected, it is known that a small percentage of cells may integrate the foreign DNA into their genome, depending on the expression vector and transfection technique used. To identify and select these integrants, a selectable marker (e.g., resistance to antibiotics) is typically introduced into the host cells along with the gene of interest. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have integrated the selectable marker gene survive, while other cells die), among other methods known in the art.

[0098] I. Formulation and Cell Culture In certain embodiments, the cells of the present disclosure may be specially formulated and / or cultured in a particular medium. The cells may be formulated in such a way that they are suitable for delivery to a recipient without adverse effects.

[0099] In a particular embodiment, the medium may be prepared using a medium used for culturing animal cells as the basal medium, such as, for example, AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, αMEM, DMEM, Ham, RPMI-1640, Fischer's medium, and any combination thereof, but is not limited thereto, as long as the medium can be used for culturing animal cells. In particular, the medium may be xeno-free or chemically defined.

[0100] The medium can be a serum-containing medium, a serum-free medium, or a xeno-free medium. From the viewpoint of preventing contamination with components derived from different animals, serum derived from the same animal as the stem cells can be used. Serum-free medium refers to a medium that does not contain untreated or unpurified serum, and therefore can include media containing purified blood-derived components or animal tissue-derived components (e.g., growth factors).

[0101] The medium may or may not contain a serum replacement. Serum replacements may include materials that suitably contain albumin (such as lipid-rich albumin, bovine albumin, recombinant albumin or humanized albumin, plant starch, dextrans and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, or equivalents thereof. Serum replacements may be prepared, for example, by methods disclosed in WO 98 / 30679, which is incorporated herein in its entirety. Alternatively, more conveniently, any commercially available material may be used. Commercially available materials include Knockout Serum Replacement (KSR), Chemically Defined Lipid Concentrate (Gibco), Glutamax (Gibco), and the like.

[0102] In certain embodiments, the medium may contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more of the following: vitamins such as biotin, DL-α-tocopherol acetate, DL-α-tocopherol, vitamin A (acetate); proteins such as BSA (bovine serum albumin) or human albumin, fatty acid free fraction V; catalase; human recombinant insulin; human transferrin; superoxide dismutase; other components such as corticosterone; D-galactose; ethanolamine HCl; glutathione (reduced); L-carnitine HCl; linoleic acid; linolenic acid; progesterone; putrescine diHCl; sodium selenite; and / or T3 (triode-I-thyronine). In certain embodiments, one or more of these may be specifically excluded.

[0103] In some embodiments, the medium further comprises one or more vitamins. In some embodiments, the medium comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen of the following (and any range derivable therein): biotin, DL α-tocopherol acetate, DL α-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or the medium comprises a combination thereof or a salt thereof. In some embodiments, the medium comprises or consists essentially of biotin, DL α-tocopherol acetate, DL α-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, and vitamin B12. In some embodiments, the vitamin comprises or consists essentially of biotin, DL α-tocopherol acetate, DL α-tocopherol, vitamin A, or combinations or salts thereof. In some embodiments, the medium further comprises a protein. In some embodiments, the protein comprises albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. In some embodiments, the medium further comprises one or more selected from corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triode-I-thyronine, or combinations thereof. In some embodiments, the medium comprises one or more of B-27® supplement, Xenofree B-27® supplement, GS21™ supplement, or combinations thereof. In some embodiments, the medium comprises or further comprises amino acids, monosaccharides, inorganic ions. In some embodiments, the amino acids include arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or a combination thereof.In some embodiments, the inorganic ions include sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof. In some embodiments, the medium further includes one or more of molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof. In certain embodiments, the medium comprises or consists essentially of one or more vitamins discussed herein, and / or one or more proteins discussed herein, and / or one or more of corticosterone, D-galactose, ethanolamine, glutathione, L-carnitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triode-I-thyronine, B-27® supplement, XenoFree B-27® supplement, GS21™ supplement, amino acids (such as, for example, arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharides, inorganic ions (such as sodium, potassium, calcium, magnesium, nitrogen, and / or phosphorus) or salts thereof, and / or molybdenum, vanadium, iron, zinc, selenium, copper, or manganese. In certain embodiments, one or more of these may be specifically excluded.

[0104] The medium may also include one or more exogenously added fatty acids or lipids, amino acids (such as non-essential amino acids), vitamin(s), growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, and / or inorganic salts, one or more of which may be specifically excluded in certain embodiments.

[0105] One or more of the media components can be added at a concentration of at least, at most, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng / L, ng / ml, μg / ml, mg / ml, or any range derivable therein.

[0106] In certain embodiments, the cells of the present disclosure are specially formulated. They may or may not be formulated as a cell suspension. In certain cases, they are formulated in a single dose form. They may be formulated for systemic or local administration. In some cases, the cells are formulated for storage prior to use, and the cell formulation may include one or more cryopreservatives, such as DMSO (e.g., in 5% DMSO). The cell formulation may include albumin, including human albumin, and a specific formulation includes 2.5% human albumin. The cells may be specially formulated for intravenous administration, for example, formulated for intravenous administration in less than 1 hour. In certain embodiments, the cells are in a formulated cell suspension that is stable for 1, 2, 3, 4 hours or more from the time of thawing at room temperature.

[0107] V. Administration of the Compositions In one embodiment of the present disclosure, an effective amount of fibroblasts as prepared by the method encompassed in the present disclosure is administered to an individual for the treatment or prevention of one or more disease conditions.In a specific embodiment, fibroblasts are administered to improve endothelial responsiveness, increase immune cell activity, cell differentiation, and reverse splenic atrophy.

[0108] In some embodiments, encapsulated RNA, microRNA, siRNA, or other RNAi compositions are administered along with the cell population, which may stimulate tissue regeneration locally or reverse tissue involution.

[0109] An embodiment of the present disclosure provides a method for co-administration of universal donor fibroblasts with one or more agents that stimulate immune cell activity in the spleen [9, 10]. In a specific embodiment of the present disclosure, a method for co-administration of universal donor fibroblasts with one or a combination of growth factors, chemokines, and / or cytokines is provided. In one embodiment of the present disclosure, universal donor fibroblasts derived from fibroblasts treated under conditions that reduce immunogenicity are utilized to stimulate vascular endothelial growth factor (VEGF) production by introduced modified / unmodified fibroblasts or from endogenous cells under the regulatory control of the individual's nerve growth factor (NGF), thereby leading to tissue regeneration and revascularization of the spleen, as has been demonstrated in other organs such as the thymus [11-13].

[0110] Therapies provided herein may include administering therapeutic compositions (e.g., fibroblasts, immune cells, derivatives of cells, exosomes from fibroblasts, drugs, etc.), alone or in combination. Therapeutic agents can be administered in any suitable manner known in the art. For example, the first and second therapies can be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the first and second treatments are administered in separate compositions. In some embodiments, the first and second treatments are in the same composition.

[0111] The embodiments of the present disclosure relate to compositions and methods, including therapeutic compositions. Different therapies may be administered in one composition or in two or more compositions, such as two compositions, three compositions, or four compositions. Various combinations of compositions may be employed.

[0112] The therapeutic compositions of the present disclosure (e.g., cells, cell products, including or derived from fibroblasts) may be administered by the same or different routes of administration. In some embodiments, the therapy is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, implanted, inhaled, intrathecally, intraventricularly, or intranasally. In some embodiments, the composition is administered directly to the spleen. In some embodiments, the antibiotic is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, implanted, inhaled, intrathecally, intraventricularly, or intranasally. The appropriate dosage may be determined based on the type of disease being treated, the severity and course of the disease, the individual's clinical condition, the individual's clinical history and response to treatment, and the discretion of the attending physician.

[0113] Therapeutic agents include various "unit doses." A unit dose is defined as containing a predetermined amount of a therapeutic composition. The amount administered, as well as the particular route and formulation, are within the discretion of those skilled in the clinical art. A unit dose need not be administered as a single injection, but may include continuous infusions over a period of time. In some embodiments, a unit dose comprises a single administrable dose.

[0114] The dosage depends on the desired therapeutic effect, both in number of treatments and unit dosage. Effective amount is understood to refer to the amount required to achieve a specific effect. In the implementation of certain embodiments, it is contemplated that a dosage ranging from 10 mg / kg to 200 mg / kg can affect the protective capacity of these agents. Thus, dosages include about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 μg / kg, mg / kg, μg / day, or mg / day, or any range derivable therein. Furthermore, such dosages can be administered multiple times during the day and / or multiple days, weeks, or months.

[0115] In certain embodiments, an effective amount of the pharmaceutical composition is one that can provide a blood level of about 1 μM to 150 μM. In other embodiments, an effective amount provides a blood level of about 4 μM to 100 μM; or about 1 μM to 100 μM; or about 1 μM to 50 μM; or about 1 μM to 40 μM; or about 1 μM to 30 μM; or about 1 μM to 20 μM; or about 1 μM to 10 μM; or about 10 μM to 150 μM; or about 10 μM to 100 μM; or about 10 μM to about 50 μM; or about 25 μM to about 150 μM; or about 25 μM to about 100 μM; or about 25 μM to about 50 μM; or about 50 μM to about 150 μM; or about 50 μM to about 100 μM (or any range derivable therein). In other embodiments, the dose may provide the following blood levels of drug resulting from the therapeutic agent being administered to a subject: about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μM, or any range derivable therein. In certain embodiments, a therapeutic agent administered to a subject is metabolized in the body to a metabolic therapeutic agent, in which case blood levels may refer to the amount of the therapeutic agent. Alternatively, to the extent that a therapeutic agent is not metabolized by the subject, blood levels discussed herein may refer to the therapeutic agent that is not metabolized.

[0116] Precise amounts of a therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting the dosage include the physical and clinical condition of the patient, the route of administration, the intended therapeutic goal (palliation of symptoms versus cure), and the efficacy, stability, and toxicity of the particular therapeutic agent or other therapy the subject may be undergoing.

[0117] Those skilled in the art will understand and appreciate that dosage units of μg / kg or mg / kg of body weight can be converted and expressed in equivalent concentration units of μg / ml or mM (blood concentration). It is also understood that uptake is species and organ / tissue dependent. Applicable conversion factors and physiological assumptions regarding uptake and concentration measurements are well known, and one skilled in the art will be able to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacy and results described herein.

[0118] In some embodiments, about 10 per 100 kg 5 ~about 10 13 of cells are administered to a human per infusion. In some embodiments, about 1.5×10 cells per 100 kg. 6 ~Approx. 1.5×10 12 In some embodiments, about 1×10 cells per 100 kg are injected. 9 ~Approx. 5×10 11 In some embodiments, about 4×10 cells per 100 kg are injected. 9 ~Approx. 2×10 11 In some embodiments, about 5×10 cells per 100 kg are injected. 8 cells ~ approx. 1×10 11 In some embodiments, a single dose of cells is provided. In some embodiments, multiple doses are provided. In some embodiments, multiple doses are provided over 3-7 consecutive days. In some embodiments, 3-7 doses are provided over 3-7 consecutive days. In some embodiments, 5 doses are provided over 5 consecutive days. In some embodiments, about 10 per 100 kg of cells are administered. 5 ~about 10 13 In some embodiments, a single dose of cells of about 1.5×10 per 100 kg is provided. 8 ~Approx. 1.5×10 12 In some embodiments, a single dose of cells of about 1×10 per 100 kg is provided. 9 ~Approx. 5×10 11In some embodiments, a single dose of cells of about 5×10 per 100 kg is provided. 10 A single dose of cells is provided. In some embodiments, 1×10 per 100 kg 10 A single dose of cells is provided. In some embodiments, about 10 per 100 kg 5 ~ to about 10 13 In some embodiments, multiple administrations of cells of about 1.5×10 per 100 kg are provided. 8 ~Approx. 1.5×10 12 In some embodiments, multiple administrations of cells of about 1×10 per 100 kg are provided. 9 ~Approx. 5×10 11 Multiple doses of cells are provided over 3-7 consecutive days. In some embodiments, about 4×10 cells per 100 kg are administered. 9 Multiple doses of cells are provided over 3-7 consecutive days. In some embodiments, about 2×10 cells per 100 kg are administered. 11 Multiple doses of cells are provided over 3-7 consecutive days. In some embodiments, about 3.5×10 9 Five doses of cells of about 4×10 are provided over five consecutive days. 9 In some embodiments, five doses of cells of about 1.3×10 are provided over five consecutive days. 11 Five doses of cells are provided over five consecutive days. In some embodiments, about 2×10 11 Five doses of cells are provided over five consecutive days.

[0119] VI. Kits of the Disclosure Any of the cellular and / or non-cellular compositions described herein, or similar thereto, may be included in the kit. In a non-limiting example, one or more reagents for use in a method for preparing fibroblasts, fibroblast-derived products, or derivatives thereof (e.g., fibroblast-derived exosomes) may be included in the kit. Such reagents include cells, vectors, one or more growth factors, vectors, one or more co-stimulatory factors, media, enzymes, buffers, nucleotides, salts, primers, compounds, etc. The components of the kit are provided in suitable containers.

[0120] Some components of the kit may be packaged in aqueous media or in lyophilized form. The container means of the kit generally includes at least one vial, test tube, flask, bottle, syringe or other container means into which the components may be placed and preferably suitably dispensed. If more than one component is included in the kit, the kit generally includes a second, third or other additional container into which the additional components may be placed separately. However, various combinations of components may be included in the vial. The kit of the present disclosure also generally includes a means for containing the components in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.

[0121] When the components of the kit are provided in one and / or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly useful. In some cases, the container means may itself be a syringe, pipette, and / or other such device, or may be a substrate having multiple compartments for the desired reaction.

[0122] Some components of the kit may be provided as a dry powder. When reagents and / or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may be provided in another container means. The kit may also comprise a second container means for containing a sterile acceptable buffer and / or other diluent.

[0123] In specific embodiments, the reagents and materials include primers, nucleotides, appropriate buffers or buffering reagents, salts, etc. for amplifying the desired sequence, and in some cases, the reagents include equipment or reagents for isolating specific desired cells.

[0124] In certain embodiments, one or more instruments suitable for extracting one or more samples from an individual are present in the kit. The instruments may be syringes, fine needles, scalpels, etc.

[0125] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the design as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the processes, machines, manufacture, compositions of matter, means, methods and steps described herein. As will be readily understood by those skilled in the art from the present disclosure, any currently existing or later developed processes, machines, manufacture, compositions of matter, means, methods or steps that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized in accordance with the present disclosure. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods or steps.

[0126] All methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present disclosure have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the methods in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the present disclosure. More specifically, it will be apparent that certain agents that are chemically and physiologically related may be substituted for the agents described herein with the same or similar results. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present disclosure as defined by the appended claims.

[0127] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the present invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the processes, machines, manufacture, compositions of matter, means, methods and steps described herein. As will be readily understood by those skilled in the art from the disclosure of the present disclosure, any currently existing or later developed process, machine, manufacture, composition of matter, means, method or step that performs substantially the same function or achieves substantially the same result as the corresponding embodiment described herein can be utilized in accordance with the present disclosure. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods or steps.

[0128] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. 1. Clark, R.A. and T.S. Kupper, IL-15 and dermal fibroblasts induce proliferation of natural regulatory T cells isolated from human skin. Blood, 2007. 109(1): p. 194-202. 2. Quandelacy, T.M., et al., Age- and sex-related risk factors for influenza-associated mortality in the United States between 1997-2007. Am J Epidemiol, 2014. 179(2): p. 156-67. 3. Tan, J.K. and T. Watanabe, Murine spleen tissue regeneration from neonatal spleen capsule requires lymphotoxin priming of stromal cells. J Immunol, 2014. 193(3): p. 1194-203. 4. Tan, J.K.H. and T. Watanabe, Determinants of postnatal spleen tissue regeneration and organogenesis. NPJ Regen Med, 2018. 3: p. 1. 5. Turner, V.M. and N.A. Mabbott, Influence of ageing on the microarchitecture of the spleen and lymph nodes. Biogerontology, 2017. 18(5): p. 723-738. 6. Sun, L., et al., FSP1(+) fibroblast subpopulation is essential for the maintenance and regeneration of medullary thymic epithelial cells. Sci Rep, 2015. 5: p. 14871. 7. Riera, M., S. Buczacki, and Z.A. Khan, Splenic regeneration following splenectomy and impact on sepsis: a clinical review. J R Soc Med, 2009. 102(4): p. 139-42. 8. Han, D., et al., Targeting Brain-spleen Crosstalk After Stroke: New Insights Into Stroke Pathology and Treatment. Curr Neuropharmacol, 2021. 19(9): p. 1590-1605. 9. Bronte, V. and M.J. Pittet, The spleen in local and systemic regulation of immunity. Immunity, 2013. 39(5): p. 806-18. 10. Lewis, S.M., A. Williams, and S.C. Eisenbarth, Structure and function of the immune system in the spleen. Sci Immunol, 2019. 4(33). 11. Park, H.J., et al., Up-regulation of VEGF expression by NGF that enhances reparative angiogenesis during thymic regeneration in adult rat. Biochim Biophys Acta, 2007. 1773(9): p. 1462-72. 12. Zanardo TE, C., et al., Decellularized Splenic Matrix as a Scaffold for Spleen Bioengineering. Front Bioeng Biotechnol, 2020. 8: p. 573461. 13. Smith, J.N., et al., 15-PGDH inhibition activates the splenic niche to promote hematopoietic regeneration. JCI Insight, 2021. 6(6). 14. Lim, H.K. and H.C. O'Neill, Identification of Stromal Cells in Spleen Which Support Myelopoiesis. Front Cell Dev Biol, 2019. 7: p. 1. 15. Wang, Z., et al., The spleen may be an important target of stem cell therapy for stroke. J Neuroinflammation, 2019. 16(1): p. 20. 16. Faustman, D.L., Regenerative medicine: Stem cell research turns to the spleen. Discov Med, 2005. 5(29): p. 447-9. 17. Faustman, D.L. and M. Davis, Stem cells in the spleen: therapeutic potential for Sjogren's syndrome, type I diabetes, and other disorders. Int J Biochem Cell Biol, 2010. 42(10): p. 1576-9.

Claims

1. 1. A method of regenerating the spleen or splenic tissue of an individual comprising administering to the individual an effective amount of a cell population, thereby regenerating the splenic tissue of the individual, wherein the cell population comprises unmodified or modified fibroblasts, unmodified or modified immune cells, activated or non-activated fibroblasts, activated or non-activated immune cells, fibroblast derivatives, immune cell derivatives, or combinations thereof.

2. The method of claim 1, wherein the fibroblast derivative comprises extracellular vesicles, exosomes, microvesicles, apoptotic bodies, or other fragments or biological components of fibroblasts.

3. 3. The method of claim 1 or 2, wherein the population of cells is activated by exposing the cells to one or more agents capable of activating the cells prior to and / or during administration.

4. 4. The method of claim 3, wherein the agent comprises one or more nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, hormones, or combinations thereof.

5. The method of any one of claims 1 to 4, wherein the population of cells is activated in vitro, in vivo, or ex vivo.

6. The method of any one of claims 1 to 5, wherein the cell population is autologous, allogeneic, xenogeneic, syngeneic, or a combination thereof to the individual.

7. The method of any one of claims 1 to 6, further comprising administering to the individual one or more adjuvants.

8. 8. The method of claim 7, wherein the adjuvant comprises a chemical adjuvant, a viral adjuvant, a bacterial adjuvant, or a combination thereof.

9. 9. The method of claim 7 or 8, wherein the adjuvant comprises encapsulated RNA, microRNA, siRNA, or a combination thereof.

10. The method according to any one of claims 1 to 9, wherein regeneration comprises organogenesis and / or T cell development and / or B cell development.

11. The method of any one of claims 1 to 10, wherein regeneration comprises reversal of splenic atrophy.

12. The method of any one of claims 1 to 11, wherein regeneration comprises reactivating or stimulating the production of immune cells in the spleen.

13. The method according to any one of claims 1 to 12, wherein the regeneration comprises splenic differentiation.

14. The method according to any one of claims 1 to 13, wherein the regeneration comprises recruitment of the spleen, differentiation of stem cells into splenic epithelial cells and / or splenic fibroblasts, and / or expansion of splenic epithelial cells and / or splenic fibroblasts.

15. The method of claim 14 , wherein the splenic fibroblasts comprise splenic reticular fibroblasts.

16. The method of any one of claims 1 to 15, wherein the activated cell population is administered systemically to the individual.

17. The method of any one of claims 1 to 15, wherein the activated cell population is administered locally to the spleen of the individual.

18. 1. An in vitro method of producing activated or non-activated fibroblasts, derivatives of activated fibroblasts, activated or non-activated immune cells, derivatives of activated or non-activated immune cells, or combinations thereof, comprising exposing fibroblasts and / or immune cells to one or more conditions that induce activation, wherein the activated fibroblasts, derivatives of activated fibroblasts, activated immune cells, and / or derivatives of activated immune cells comprise activity for regenerating spleen tissue.

19. 19. The method of claim 18, wherein the activated or non-activated fibroblasts and / or the activated or non-activated immune cells are autologous to the spleen.

20. 19. The method of claim 18, wherein the activated or non-activated fibroblasts and / or the activated or non-activated immune cells are allogeneic to the spleen.

21. The method of claim 18, wherein the activated fibroblasts and / or activated immune cells are xenogeneic to the spleen.

22. 20. The method of claim 18, wherein the activated fibroblasts and / or activated immune cells are syngeneic to the spleen.

23. The method of any one of claims 18 to 22, wherein regeneration comprises stimulating, improving and / or increasing immune cell production and / or activity in the spleen.

24. 22. The method of any one of claims 18 to 21, wherein the one or more conditions comprise one or more nucleic acids, cytokines, chemokines, transcription factors, epigenetic factors, growth factors, hormones, or combinations thereof.

25. A method of stimulating, improving, or increasing the production and / or activity of cells in the spleen in an individual, comprising providing to the individual an effective amount of activated fibroblasts, derivatives of activated fibroblasts, activated immune cells, derivatives of activated immune cells, or a combination thereof.

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