Mesenchymal progenitor cells for enhancing partial reprogramming of target cells

Abstract

Provided herein are methods and related compositions for enhancing partial reprogramming of target cells in a subject in need thereof (e.g., a human subject suffering from or at risk of suffering from a disease), the methods comprising administering to the subject a plurality of mesenchymal mass spectrum progenitor cells or stem cells (MLPSC), exosomes derived therefrom, or conditioned media derived therefrom, the subject expresses or will express one or more reprogramming factors in a population of target cells, thereby causing a plurality of the target cells in the subject to be partially reprogrammed but not fully reprogrammed.

Description

[0001] This application claims priority to AU 2022901577 filed on June 8, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention relates to methods and related compositions for enhancing partial reprogramming and their therapeutic applications. Background Art

[0003] When experimentally induced to express one or more "reprogramming factors", a variety of differentiated mammalian cell types can regain a wide range of developmental potential. When such reprogramming factors are expressed in differentiated cells for a sufficient period of time, differentiated cells are reprogrammed into pluripotent stem cells called "induced pluripotent stem cells" (iPSC). One of the most significant features of the reprogramming process may be that in addition to "resetting" cell fate, cell "age" (relative to cell donor age) is also reset, thereby producing regenerative cells, as reflected by a series of features (such as telomere length, epigenetic changes and gene expression changes). In fact, human iPSC systems can be generated from young to old individuals, and the hiPSC age determined from these systems is actually equivalent to embryonic age.

[0004] It has been demonstrated that during the cell reprogramming process, the resetting of cell age occurs before the loss of differentiated cell identity. Therefore, by limiting the reprogramming factor expression time, differentiated cells regenerate without losing cell type identity, and this process is called "partial reprogramming". Various health conditions are related to damaged and / or aging tissues (such as cardiovascular disease, low back pain and osteoarthritis). Although partial reprogramming of cells in vivo has been achieved in animal models, the extremely low efficiency of the process is a serious obstacle to the potential treatment of partial reprogramming. Therefore, there is a continuous need for methods and compositions for enhancing partial reprogramming. Summary of the invention

[0005] Mesenchymal lineage progenitor cells or stem cells (MLPSC) (such as human mesenchymal stem cells (hMSC) and human mesenchymal progenitor cells (hMPC)) have a very wide range of therapeutic properties and uses. Among them are its significant anti-inflammatory properties, for example, its ability to downregulate proinflammatory cytokine levels and regulate immune cell responses, for example, downregulating mTOR activity in T cells and effector functions in natural killer (NK) cells. Although it is not desired to be bound by theory, it is believed that several cell signaling pathways and immune cell effector functions downregulated by MLPSC are pathways and effector functions that inhibit and reduce the efficiency of cell reprogramming (including partial reprogramming), and may be particularly important in the context of partial reprogramming of elderly subjects in which proinflammatory environments are found in many tissues. In addition, in some embodiments, MLPSC can suppress immune responses to expression vectors (such as recombinant viruses), which can be used to deliver reprogramming factors in vivo or induce their expression in target cell populations. In addition, some therapeutic effects provided by MLPSC can also be provided by exosomes separated from MLPSC. Therefore, it is believed that in the presence of MLPSCs or exosomes derived from MLPSCs, the efficiency of cell reprogramming will be enhanced.

[0006] Thus, in one aspect, provided herein is a method for enhancing partial reprogramming of target cells in a subject in need thereof, the method comprising administering to the subject a plurality of mesenchymal lineage progenitor or stem cells (MLPSCs), exosomes derived therefrom, or conditioned medium derived from a culture of a plurality of MLPSCs that express or will express one or more reprogramming factors in a target cell population upon administration, thereby causing a plurality of target cells in the subject to be partially reprogrammed.

[0007] In some embodiments, the target cell population comprises one or more exogenous nucleic acids encoding one or more reprogramming factors.

[0008] In other embodiments, the target cell population comprises one or more exogenous nucleic acids encoding one or more targeted transactivators for inducing endogenous expression of one or more reprogramming factors.

[0009] In some embodiments of the methods described herein, the MLPSCs are derived from human STRO-1 + or TNAP +Mesenchymal progenitor cell (MPC) group or cell culture group expanded from human mesenchymal stem cell (hMSC) group. In some embodiments, MLPSC is a pre-licensed MLPSC. In some embodiments, in the case where MLPSC is a pre-licensed MLPSC, the pre-licensed MLPSC is a MLPSC cultured and expanded in a culture medium containing the following components: IFN-γ and / or TNF-α; and one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1-α, MIP-1-β and IP-10. In some embodiments, the culture medium contains serum, the serum contains IFN-γ and / or TNF-α; and one or more of the above-mentioned pro-inflammatory cytokines. In some embodiments, in the case where the culture medium contains serum, the culture medium contains newborn mammalian serum. In some embodiments, newborn mammalian serum is newborn calf serum.

[0010] In some embodiments, the MLPSC is a MLPSC genetically modified to overexpress indoleamine 2,3-dioxygenase (IDO) and / or cyclooxygenase-2 (COX-2).

[0011] In some embodiments, the method comprises administering a plurality of MLPSCs. In other embodiments, the method comprises administering exosomes derived from a plurality of MLPSCs. In other embodiments, the method comprises administering conditioned medium derived from a culture of a plurality of MLPSCs.

[0012] In some embodiments, the subject to be administered MLPSCs, exosomes thereof, or culture medium suffers from a health condition to be treated selected from the group consisting of cardiovascular disease, vascular endothelial disorders, low back pain, inflammatory diseases, osteoarthritis, metabolic diseases, kidney disease, liver disease, and neurological disorders. In some embodiments, the health condition is cardiovascular disease, low back pain, or inflammatory disease.

[0013] In some embodiments, the target cells to be partially reprogrammed in the subject are selected from the group consisting of cardiomyocytes, nucleus pulposus cells, kidney cells, chondrocytes, hepatocytes, endothelial progenitor cells, chondroprogenitor cells, neurons, and glial cells.

[0014] In some embodiments of the methods disclosed herein, one or more reprogramming factors are selected from the group consisting of: OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG. In some embodiments, one or more reprogramming factors include OCT4, SOX2 and KLF4. In other embodiments, one or more reprogramming factors include OCT4, SOX2, KLF4 and c-MYC. In some embodiments, one or more reprogramming factors include OCT4, SOX2 and KLF4 but do not include c-MYC. In other embodiments, one or more reprogramming factors: (i) do not include LIN 28; or (ii) do not include NANOG. In some embodiments, one or more reprogramming factors include OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG. In other embodiments, the one or more reprogramming factors consist of: (i) OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG; (ii) OCT4, SOX2, KLF4, and c-MYC; or (iii) OCT4, SOX2, and KLF4.

[0015] In some embodiments, the one or more exogenous nucleic acids in the target cell include one or more synthetic mRNAs encoding one or more reprogramming factors or encoding one or more targeted transactivators. In some embodiments, the one or more synthetic mRNAs include one or more nucleoside-modified mRNAs. In other embodiments, the one or more synthetic mRNAs are unmodified synthetic mRNAs.

[0016] In some embodiments, one or more exogenous nucleic acids include one or more non-integrated expression cassettes for expressing one or more encoded reprogramming factors or targeted transactivators in mammalian cells. In some embodiments, in the case where one or more exogenous nucleic acids include one or more non-integrated expression cassettes, one or more non-integrated expression cassettes are provided as one or more of the following: plasmid expression vectors, minicircle expression vectors, linear amplicon, circular DNA amplicon, recombinant DNA viral genome or amplicon or recombinant RNA viral genome or amplicon. In some embodiments, in the case of using a non-integrated expression cassette, at least one of the one or more encoded reprogramming factors or targeted transactivators is controlled by a target cell selective promoter. In some embodiments, the target cell selective promoter is selected from the group consisting of: cardiomyocyte selective promoter, nucleus pulposus cell selective promoter, renal cell selective promoter, hepatocyte selective promoter, endothelial progenitor cell selective promoter, chondroprogenitor cell selective promoter, glial cell selective promoter and neuron selective promoter.

[0017] In some embodiments of any of the aforementioned methods, at least one exogenous nucleic acid is a polycistronic.

[0018] In some embodiments, expression of one or more reprogramming factors is inducible and reversible. In some embodiments, the inducible and reversible expression is regulated by reverse tetracycline transactivator (rtTA).

[0019] In some embodiments, the MLPSCs to be administered are modified MLPSCs comprising at least one of the following: an exogenous anti-inflammatory agent: miRNA, antagomiR, siRNA, RNAi, antisense oligonucleotide, or antisense RNA. In some embodiments, the MLPSCs are modified MLPSCs comprising an anti-p16 INK4a At least one of a miRNA, antagomiR, siRNA, RNAi, antisense oligonucleotide, or antisense RNA that targets at least one of the mTOR signaling pathway, or the c-Jun N-terminal kinase (JNK) signaling pathway.

[0020] In some embodiments, one or more reprogramming factors are expressed continuously for at least two days but no longer than about 15 days.

[0021] In some embodiments, multiple MLPSCs are administered to a subject after expression of one or more reprogramming factors has begun. In other embodiments, multiple MLPSCs are administered to a subject before expression of one or more reprogramming factors has begun. In other embodiments, multiple MLPSCs are administered while initiating expression of reprogramming factors.

[0022] In some embodiments of any of the foregoing methods, the method further comprises the step of delivering one or more exogenous nucleic acids to the target cell population.

[0023] In some embodiments of any of the foregoing methods, the method further comprises administering prostaglandin E2 to the subject. In some embodiments, the method further comprises administering a blocking antibody to the natural killer cell receptor NKG2D.

[0024] In a related aspect, provided herein is a method for enhancing partial reprogramming of target cells in a postnatal subject in need thereof, the method comprising administering to the subject a plurality of MLPSCs, exosomes derived therefrom, or conditioned medium derived from a culture of the plurality of MLPSCs, wherein the subject expresses exogenous reprogramming factors comprising OCT4, SOX2, KLF4, and c-MYC in a target cell population simultaneously with or after administration, wherein one or more exogenous nucleic acids for expressing the exogenous reprogramming factors have been delivered to the target cell population (i) as one or more synthetic mRNAs; (ii) by one or more non-integrating recombinant viruses; or (iii) as a combination of (i) and (ii); thereby a plurality of the target cells in the subject are partially reprogrammed.

[0025] In some embodiments, one or more exogenous nucleic acids are delivered to a target cell population as one or more synthetic mRNAs.

[0026] In other embodiments, one or more exogenous nucleic acids are delivered to the target cell population via one or more non-integrating recombinant viruses. In some embodiments, the non-integrating recombinant virus is selected from the group consisting of: adenovirus, adeno-associated virus, non-integrating lentivirus, human cytomegalovirus (CMV), herpes simplex virus (HSV) and Sendai virus.

[0027] In some embodiments, MLPSCs are administered simultaneously with the expression of exogenous reprogramming factors. In other embodiments, MLPSCs are administered prior to the expression of exogenous reprogramming factors.

[0028] In some embodiments, the method further comprises delivering one or more exogenous nucleic acids to the target cell population to express the exogenous reprogramming factors.

[0029] In some embodiments, the MLPSC is a pre-licensed MLPSC. In some embodiments, in the case where the MLPSC is a pre-licensed MLPSC, the pre-licensed MLPSC is an MLPSC cultured and expanded in a culture medium containing the following ingredients: IFN-γ and / or TNF-α; and one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1-α, MIP-1-β, and IP-10. In some embodiments, the culture medium contains serum containing IFN-γ and / or TNF-α; and one or more of the above-mentioned pro-inflammatory cytokines. In some embodiments, in the case where the culture medium contains serum, the culture medium contains newborn mammalian serum. In some embodiments, the newborn mammalian serum is newborn calf serum.

[0030] In a related aspect, provided herein is a population of MLPSCs when used in any of the foregoing methods as disclosed herein.

[0031] In another aspect, the present invention provides a composition for enhancing partial reprogramming of target cells, comprising a plurality of modified mesenchymal lineage progenitor or stem cells (MLPSCs), wherein the MLPSCs comprise one or more exogenous nucleic acids encoding one or more of the following: (a) reprogramming factors; or (b) targeted transactivators that induce expression of one or more endogenous reprogramming factors, wherein: (i) when one or more target cells are present, the modified MLPSCs deliver the one or more exogenous nucleic acids to the one or more target cells; (ii) the one or more encoded reprogramming factors are expressed in the one or more target cells at a level sufficient to partially reprogram the one or more target cells; and (iii) the one or more encoded reprogramming factors or targeted transactivators are substantially unable to be expressed in the modified MLPSCs.

[0032] In a further aspect, provided herein is a composition or kit for enhancing partial reprogramming of a target cell, comprising:

[0033] (i) culture-expanded human mesenchymal stem cell (hMSC) populations;

[0034] (ii) a conditional replication helper virus; and

[0035] (iii) one or more helper-dependent viruses, which: (a) contain one or more expression cassettes of one or more reprogramming factors or targeted transactivators that induce expression of one or more endogenous reprogramming factors; and (b) are substantially free of viral coding sequences.

[0036] In some embodiments, hMSCs are transduced using (ii), (iii), or both (ii) and (iii). In some embodiments, one or more expression cassettes are inducible and reversible expression cassettes. In some embodiments, the conditionally replicating helper virus or one or more helper-dependent viruses encode a regulatable transactivator (e.g., rtTA) that controls the expression of one or more inducible and reversible expression cassettes.

[0037] In some embodiments, the conditional replication helper virus and the one or more helper-dependent viruses are adenoviruses, herpes simplex virus (HSV), non-integrating lentiviruses, or adeno-associated viruses (AAV). In some embodiments, the conditional replication helper virus and the one or more helper-dependent viruses are adenoviruses.

[0038] In some embodiments, the culture-expanded hMSCs are pre-permitted hMSCs.

[0039] In some embodiments, the composition is provided as a pharmaceutical composition comprising a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises one or more of prostaglandin E2 or a blocking antibody against natural killer cell receptor NKG2D.

[0040] In a related aspect, a method for enhancing partial reprogramming of target cells is provided herein, comprising administering to a subject in need thereof any of the compositions just mentioned comprising a conditionally replicating virus and a helper-dependent virus. In some embodiments, the subject suffers from or is at risk of suffering from a health condition selected from the group consisting of: cardiovascular disease, vascular endothelial disorders, low back pain, inflammatory diseases, osteoarthritis, metabolic diseases, kidney disease, liver disease, and neurological disorders. In some embodiments, the transduced hMSCs are administered by a local administration route. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1: Evaluation and comparison of serum cytokine levels. 1:1 FCS / NBCS (serum A); fetal bovine serum (serum B); FBS from different suppliers (serum C). Panels A to I show the measured values ​​of cytokines IFN-γ, IL-6, IL-8 and IL-17A, MCP-1, MIP-1α, MIF-1β, IP-10, and TNF-α, respectively.

[0042] Figure 2 : Analysis of changes from baseline in cardiac echo parameters at 12 months - All subjects using pre-permitted MPCs cultured in medium containing 10% FCS versus 5% FCS / 5% NBCS.

[0043] Figure 3 : Analysis of changes from baseline in Echo parameters at 12 months - subjects with persistent inflammation (hsCRP ≥ 2).

[0044] Figure 4 : Analysis of changes from baseline in Echo parameters at 12 months - subjects without persistent inflammation (hsCRP<2).

[0045] Figure 5 : MPCs cultured in the presence or absence of neonatal serum resulted in CV death in subjects with persistent inflammation (hsCRP ≥ 2).

[0046] Figure 6 : MPCs cultured in the presence or absence of neonatal serum resulted in a 3-point composite MACE (MI, stroke, or CV death) in subjects with persistent inflammation (hsCRP ≥ 2).

[0047] Figure 7: 3-point composite MACE (MI, stroke or CV death) of subjects administered MPCs cultured in the presence or absence of neonatal serum at all or last passages. The LHS graph represents data from all patients (A). The RHS graph represents data from patients with persistent inflammation (CRP>2 mg / ml; (B)).

[0048] Figure 8: 3-point composite MACE (A) and terminal cardiac events (TCE; B) in subjects with the most severe disease (NTpro-BNP>1000 ng / ml; CRP>2 mg / ml) administered MPCs cultured in the presence or absence of neonatal serum at all or last passages.

[0049] Fig. 9 : Schematic diagram of the combinatorial viral reprogramming platform in hMSCs. DETAILED DESCRIPTION

[0050] Overview

[0051] Throughout this specification, references to individual steps, compositions of matter, groups of steps, or groups of compositions of matter are to be understood to cover both one and more (i.e., one or more) of those steps, compositions of matter, groups of steps, or groups of compositions of matter, unless expressly stated otherwise or the context requires otherwise.

[0052] Unless expressly stated otherwise, every embodiment described herein applies mutatis mutandis to every other embodiment.

[0053] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those explicitly described. It should be understood that the invention includes all such variations and modifications. The invention also includes all steps, features, compositions and compounds mentioned or indicated in this specification, either individually or collectively, and any and all combinations or any two or more of said steps or features.

[0054] The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for purposes of illustration only. Functionally equivalent products, compositions and methods, as described herein, are clearly within the scope of the present invention.

[0055] The present invention may be performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology. Such procedures are described, for example, in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, 2nd edition (1989), Volumes I, II and DI in their entirety; DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, in its entirety; Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed., 1984) IRL Press, Oxford, in its entirety, and especially in Gait's paper therein, pp. 1-22; Atkinson et al.'s paper, pp. 35-81; Sproat et al.'s paper, pp. 83-115; and Wu et al.'s paper, pp. 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, ed., 1985) IRL Press, Oxford, in its entirety. Press, Oxford, full text; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, full text; Perbal, B., A Practical Guide to Molecular Cloning (1984); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), full series; JF Ramalho Ortigao, "The Chemistry of Peptide Synthesis" See: Access Virtual Laboratory Web Knowledge Database (Interactiva, Germany); Sakakibara, D., Teichman, J., Lien, EL and Fenichel, RL (1976). Biochem. Biophys. Res. Commun. 73 336-342; Merrifield, RB (1963). J. Am. Chem. Soc. 85, 2149-2154; Barany, G.and Merrifield, R. B. (1979) in The Peptides (Gross, E. and Meienhofer, J. eds.), Vol. 2, pp. 1-284, Academic Press, New York. 12. Wünsch, E. ed. (1974) Synthese von Peptiden in Houben-Weyls Metoden der Organischen Chemie (Müler, E. ed.), Vol. 15, 4th edition, Parts 1 and 2, Thieme, Stuttgart; Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474; Handbook of Experimental Immunology, Volumes I-IV (Edited by DM Weir and CC Blackwell, 1986, Blackwell Scientific Publications); and Animal Cell Culture: Practical Approach, 3rd edition (Edited by John RW Masters, 2000), ISBN 0199637970, full text. .

[0056] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of stated steps or elements or integers or groups of steps or elements or integers but not the exclusion of any other steps or elements or integers or groups of elements or integers.

[0057] As used herein, the term "derived from" should be understood to indicate that the specified integer may be obtained from a particular source, but not necessarily directly from that source.

[0058] As used herein, the terms "prevent", "preventing" and "prevention" are understood to mean administering a prophylactically effective amount of cells and stopping or hindering or delaying or reducing the development of at least one symptom of a clinical disorder.

[0059] As used herein, the term "partial reprogramming" refers to the process of converting somatic cells into the epigenetic state of cells (i.e., "partially reprogrammed cells") by expressing one or more reprogramming factors in a sufficient time period, wherein the cells retain their original cell type or lineage identity, but show one or more changes indicating a younger or "rejuvenated" cell age (commonly referred to as "epigenetic age" or "eAge"), as indicated by changes in one or more parameters (including but not limited to genomic methylation, gene expression characteristics, metabolite production, and telomere length). For clarity, partially reprogrammed cells do not include cells that are reprogrammed to pluripotency or otherwise have lost their original cell lineage. The sufficient time period for expressing one or more reprogramming factors refers to the cumulative expression period of partially reprogrammed cells that are substantially free of completely reprogrammed cells as the final result of the process. In some embodiments, the cumulative reprogramming factor expression period is no more than about 21 days, preferably no more than 15 days.

[0060] As used herein, the term "full reprogramming" or "fully reprogrammed" refers to a process in which one or more reprogramming factors are expressed for a period of time long enough to lose cell identity. Typically, full reprogramming requires a cumulative reprogramming factor expression period of longer than about 21 days.

[0061] As used herein, the phrase "STRO-1 + "Pluripotent cells" should be understood to mean non-hematopoietic STRO-1 cells that are capable of forming pluripotent cell colonies. + and / or TNAP + Progenitor cells. The preferred STRO-1 is discussed in more detail herein + Pluripotent cells.

[0062] As used herein, the term "conditioned medium" as used in the context of the present disclosure refers to a culture medium obtained from MLPSC under culture conditions. Such a culture medium contains MLPSC secretomes, proteins shed from the surface of MLPSC, released metabolites, and particles such as extracellular vesicles. Therefore, the conditioned medium of the present disclosure may contain pro-angiogenic factors, such as extracellular vesicles and angiogenin, or secreted metabolites, such as prostaglandin E2. In certain instances, the present disclosure relates to extracellular vesicles, such as exosomes, obtained from a conditioned medium obtained from MLPSC under culture conditions.

[0063] As used herein, the term "subject" should be understood to mean any subject, preferably a mammal, such as a mouse, a rat, a sheep, a non-human primate, and a human. In some preferred embodiments, the subject is a human.

[0064] As used herein, the terms "treat", "treatment" and "treating" are understood to mean administering a therapeutically effective amount of cells and reducing or inhibiting at least one symptom of a clinical disorder.

[0065] As used herein, the phrase "at risk of developing" a disease indicates that one or more available clinical and / or diagnostic indicators indicate a higher than average probability of developing a particular health condition or disease.

[0066] method

[0067] Provided herein is a method for enhancing partial reprogramming of target cells in a subject in need thereof (e.g., a human subject), wherein the method comprises the step of administering to the subject a plurality of mesenchymal lineage progenitor or stem cells (MLPSCs), exosomes derived therefrom, or conditioned medium derived from a culture of MLPSCs, wherein the subject expresses or will express one or more reprogramming factors in a target cell population, whereby after a period of expression of the one or more reprogramming factors, a plurality of target cells in the subject are partially reprogrammed, wherein the length of the expression period is insufficient to completely reprogram the plurality of target cells.

[0068] In some embodiments, the target cells in the target cell population are one or more of the following cells: cardiomyocytes, nucleus pulposus cells, kidney cells, hepatocytes, endothelial progenitor cells, chondroprogenitor cells, chondrocytes, neurons and glial cells. In other embodiments, the target cells comprise skin cells, such as keratinocytes, melanocytes and fibroblasts.

[0069] In some embodiments, the target cell group includes one or more exogenous nucleic acids encoding one or more reprogramming factors. In other embodiments, the target cell group includes one or more exogenous nucleic acids, and the exogenous nucleic acids encode one or more targeting transactivators, which are used to induce the expression of endogenous genes encoding one or more reprogramming factors. In some embodiments, the method disclosed herein also encompasses the step of delivering one or more exogenous nucleic acids to the target cell group of the subject. In some preferred embodiments, the method of the present invention does not encompass the delivery step itself just mentioned.

[0070] In some embodiments, the MLPSCs are culture-expanded from a population of human STRO-1+ mesenchymal progenitor cells (MPCs). In other embodiments, the MLPCs are culture-expanded human mesenchymal stem cells (hMSCs).

[0071] In some embodiments, the MLPSC to be administered is a pre-licensed MLPSC, which refers to the MLPSC cultured and expanded in the presence of one or more pro-inflammatory stimuli, which is considered to enhance the anti-inflammatory characteristics of such MLPSC, thereby facilitating the use of MLPSC in the methods described herein (including administering MLPSC to a subject in need). In some embodiments, MLPCS is pre-licensed by culturing and expanding in a culture medium containing: IFN-γ and / or TNF-α; and one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1-α, MIP-1-β and IP-10. In some embodiments, the culture medium for the culture expansion and pre-licensing of MLPSC contains serum containing IFN-γ and / or TNF-α; and one or more of the aforementioned pro-inflammatory cytokines. In some embodiments, the culture medium contains newborn mammal serum. In some embodiments, the newborn mammal serum is newborn calf serum. In some embodiments, the culture medium contains a mixture of fetal mammal serum and newborn mammal serum. In some instances, the culture medium contains 5% (volume / volume) fetal bovine serum and 5% (volume / volume) newborn calf serum. In other instances, the culture medium contains newborn mammal serum in the range of about 5% to about 10% (volume / volume), such as 6%, 7%, 8%, 9%, or another percentage of about 5% to about 10% (volume / volume).

[0072] In some embodiments, the MLPSCs to be administered are genetically modified MLPSCs that are genetically modified to overexpress indoleamine 2,3-dioxygenase (IDO) and / or cyclooxygenase-2 (COX-2), the activities of each of which are believed to be useful in inhibiting the cytotoxic activity of NK cells, which is believed to be detrimental to partial reprogramming of target cells in vivo.

[0073] In some embodiments, MLPSCs are to be administered. In other embodiments, isolated exosomes derived from MLPSCs are to be administered. In other embodiments, conditioned medium from MLPSC cultures (e.g., pre-licensed MLPSC cultures) is to be administered.

[0074] Generally, the subject to which the target cell group needs to be partially reprogrammed is a subject suffering from a health condition to be treated by partial reprogramming of one or more target cell groups. In some embodiments, the subject suffers from a health condition selected from the group consisting of: cardiovascular disease, vascular endothelial disease, low back pain, inflammatory disease, osteoarthritis, metabolic disease, kidney disease, liver disease and neurological disorder. In some preferred embodiments, the subject is a human subject. In a preferred embodiment, the subject is a postnatal human subject. In some embodiments, the age of the postnatal human subject is about 40 to about 110 years old, such as 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 years old or about 40 to about 110 years old. Another age.

[0075] In some embodiments, the administration of MLPC, exosomes or conditioned medium is performed by a systemic administration route (intravenous or intra-arterial). In other embodiments, the administration is performed by a local administration route, preferably guided by a target tissue to be induced to partially reprogram, for example, intracardiac administration for the heart, intraarticular administration for the joint, intravertebral administration for the intervertebral disc, intracerebral administration for the brain, etc.

[0076] Reprogramming factors

[0077] Suitable reprogramming factors for the partial reprogramming methods disclosed herein include, but are not limited to, one or more of OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG. In some embodiments, the reprogramming factors include OCT4, SOX2, and KLF4. In other embodiments, the reprogramming factors include OCT4, SOX2, KLF4, and c-MYC. In some embodiments, one or more reprogramming factors include OCT4, SOX2, and KLF4 but do not include c-MYC. In some embodiments, one or more reprogramming factors: (i) do not include LIN 28; or (ii) do not include NANOG.

[0078] In some embodiments, the one or more reprogramming factors consist of: (i) OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG; (ii) OCT4, SOX2, KLF4, and c-MYC; or (iii) OCT4, SOX2, and KLF4.

[0079] In some preferred embodiments, the reprogramming factors include OCT4, SOX2, KLF4 and c-MYC.

[0080] It is also contemplated herein to use structural variants (e.g., fusion proteins, truncations, point mutations, or deletions) of any of the above reprogramming factors that have been functionally tested in vitro for cell reprogramming to pluripotency. Examples of reprogramming factor fusion proteins, such as OCT4-VP16 fusion proteins, are known in the art, such as Hammachi et al., (2012), Cell Reports, 1(2): 99-109.

[0081] In some embodiments, one or more reprogramming factors are fusion proteins comprising conditional destabilization domains (CDDs), whereby in the absence of synthetic stabilizing ligands, CDD triggers rapid degradation of fusion proteins. This system allows fine-tuning of reprogramming factor fusion protein levels by controlling the level of synthetic stabilizing ligands present. In some exemplary embodiments, CDD is dihydrofolate reductase CDD and the synthetic stabilizing ligand is trimethoprim. Examples of suitable reprogramming factor fusion proteins are known in the art, for example, in Sui et al., (2014), Stem Cell Reports, 2 (5): 721-733.

[0082] Targeted transactivators for inducing expression of any of the above-mentioned reprogramming factors from corresponding endogenous / natural genes have been described in the art. In some preferred embodiments, the targeted transactivator is a targeted transactivator based on CRISPR activation (CRISPRa). CRISPR activation (CRISPRa) uses a catalytically inactive form of Cas9 (dCas9) fused to a transactivator domain to activate transcription from an endogenous promoter. Typically, a targeted transactivator based on CRISPRa will include two components: (i) dCas9-transactivator domain fusion protein-"dCas9-tA" (e.g., VP16 transactivation domain); and (ii) a guide RNA (gRNA) that guides dCas9-tA to a specific reprogramming factor gene site in the genome to drive the expression of endogenously encoded reprogramming factors. Suitable dCas9-tAs and reprogramming factor gRNAs and methods for reprogramming cells using targeted trans-activators are known in the art, as described in, for example, Weltner et al., (2018), Nat Commun; 9:2643 and Sokka et al., (2022), Stem Cell Reports, 17(2):413-426.

[0083] In some embodiments, one or more exogenous nucleic acids include one or more synthetic mRNAs encoding one or more reprogramming factors or encoding one or more targeted transactivators. In some embodiments, one or more synthetic mRNAs include one or more nucleoside-modified mRNAs, wherein the nucleoside-modified mRNAs have RNASe resistance and / or are unlikely to induce type I interferon response. Methods for in vitro production of mRNA for reprogramming are known in the art, as reviewed in, for example, Steinle et al. (2016), Stem Cells, 35 (1): 68-79. In some embodiments, in the case of using synthetic mRNA, the synthetic mRNA includes a mixture of nucleoside-modified mRNA and non-nucleoside-modified mRNA. Kits for in vitro transcription (IVT) of modified or unmodified mRNA are commercially available, such as HyperScribe from APExBIO TM All in One mRNA Synthesis Kit (Catalog Numbers: K1063-K1068).

[0084] Methods and compositions for delivering mRNA to the whole body or specific tissues in vivo are known in the art, as reviewed in Zhang et al., (2021), Chemical Reviews, 121: 12181-12277. In some embodiments, lipid nanoparticles (LNPs) are used to deliver mRNA to subjects in the methods disclosed herein. In other embodiments, mRNA is delivered by polymer nanomicelles, as described in, for example, Chang et al., (2022), International Journal of Molecular Sciences, 23: 565. In other embodiments, particularly in the case where mRNA is directly administered to tissues containing target cells of interest (e.g., in the heart), "naked" mRNA, i.e., mRNA that is not associated with a transfection agent, is injected into a biocompatible buffer formulation (e.g., citrate-saline solution), as illustrated in Carlsson et al., (2018), Molecular Therapy Methods & Clinical Development, 9: 330-346.

[0085] In some embodiments, the one or more nucleic acids comprise one or more non-integrating expression cassettes for expressing one or more encoded reprogramming factors or targeted transactivators in mammalian cells (e.g., a target cell population in a human subject), wherein the expression cassette comprises at least a promoter operably linked to an open reading frame encoding the one or more reprogramming factors, a targeting enzyme (e.g., dCas9), or a gRNA targeting the promoter of a native gene encoding the reprogramming factor.

[0086] As used herein, the term "promoter" should be understood in its broadest context and includes transcriptional regulatory sequences (including TATA boxes or initiation elements) of genomic genes, which are required for transcription initiation in the presence or absence of additional regulatory elements (i.e., upstream activation sequences, transcription factor binding sites, enhancers, and silencers), which, for example, respond to development and / or external stimuli or change gene expression in a tissue-specific manner. In the context of the present invention, the term "promoter" is also used to describe recombinant, synthetic or fusion molecules or derivatives that confer, activate or enhance the expression of nucleic acids operably connected thereto and preferably encoding peptides or proteins. Preferably, the promoter may contain additional copies of one or more specific regulatory elements to further enhance expression and / or change the spatial expression and / or temporal expression of the nucleic acid molecule.

[0087] In the context of the present invention, a nucleic acid is "operably linked" to a promoter (i.e., under the regulatory control of a promoter) when the nucleic acid is positioned so that its expression is controlled by the promoter. A promoter is typically positioned 5' (upstream) of the nucleic acid whose expression it controls. In order to construct a heterologous promoter / nucleic acid combination, it is generally preferred that the promoter be positioned at a distance from the gene transcription start site that is approximately the same as the distance between the promoter and the gene it controls in its natural environment (i.e., the gene from which the promoter is derived). As is known in the art, some variation in this distance may be tolerated without loss of promoter function. Similarly, the preferred positioning of a regulatory sequence element relative to a heterologous nucleic acid placed under its control is defined by the positioning of the element in its natural environment (i.e., the gene from which the element is derived). Likewise, as is known in the art, some variation in this distance may also occur.

[0088] In some embodiments, the one or more non-integrating expression cassettes are provided as one or more of: a plasmid expression vector, a minicircle expression vector, a linear amplicon, a circular DNA amplicon, a recombinant DNA viral genome, an amplicon recombinant RNA viral genome, an RNA amplicon, or any combination thereof.

[0089] In some preferred embodiments, plasmid expression vectors, minicircle expression vectors, linear amplicon and circular DNA amplicon are provided as additional vectors, and the additional vectors contain a replication origin, whereby the additional vectors are replicated when their host cells undergo cell division. When the target cell populations (e.g., chondrocytes, glial cells and neural progenitor cells) to be partially reprogrammed are proliferative, additional vectors are particularly useful. Reprogramming using additional expression vectors is known in the art, for example, in Wang and Loh (2019), Cell Transplant, 28 (1 Suppl): 112S-131S and additional vectors.

[0090] In some embodiments, the non-integrating expression cassette is delivered by a recombinant virus selected from the group consisting of: adenovirus, adeno-associated virus, non-integrating lentivirus, human cytomegalovirus (CMV), herpes simplex virus (HSV) and Sendai virus. Although not wishing to be bound by theory, it is believed that administering hMLSCs shortly before or after administering the recombinant (non-integrating) virus has the additional advantage of reducing unnecessary immune neutralization of such recombinant viruses. This reduced immune neutralization leads to increased viral transduction of target cells, and ultimately to increased partial reprogramming efficiency of target cells.

[0091] In some embodiments, where one or more expression cassettes are provided as one or more DNA viral genomes, the DNA viral genomes are provided as recombinant non-integrating lentiviruses, which have very high transduction efficiencies while minimizing the risk of insertional mutagenesis. See, e.g., Gurumoorthy et al., (2022), Biomedicines, 10:107 and Abouleisa et al., (2022), Circulation, 145:1339-1355.

[0092] In other embodiments, one or more recombinant adenoviruses provide one or more expression cassettes as described, for example, in Lehmann et al., (2019), Gene Therapy, 26:432-440.

[0093] In other embodiments, one or more recombinant human cytomegaloviruses (hCMV) are used in the methods disclosed herein, which have the advantage of being able to accommodate very large inserts relative to other viral vectors. The use of recombinant CMV as a gene therapy vector has been described in the art, for example, in Jaiyan et al., (2022), Proc. Natl. Acad. Sci USA, 119(20): e2121499119.

[0094] In some embodiments, when one or more expression cassettes are provided as one or more RNA viral genomes, the RNA viral genome is provided as a recombinant RNA virus, which is substantially free of the risk of insertional mutagenesis. In some embodiments, the recombinant RNA virus to be used is measles virus (MV), which is a human negative-strand RNA paramyxovirus with a proven record of safe vaccination. Reprogramming using MV has been described, for example, in Wang et al., (2019), 26(5): 151-164.

[0095] In some embodiments, in the case of using a recombinant virus, the recombinant virus is a recombinant virus with selective tropism for target cells. For example, recombinant adeno-associated viruses (AAVs) have been developed in the art, which are engineered to have tropism for specific target cell populations after systemic administration, such as cardiomyocytes (Zincarelli et al., 2008, Mol. Ther., 16 (6): 1073-1080) and CNS neurons and / or astrocytes (Goertsen et al., 2021, Nature Neuroscience, 25: 106-115).

[0096] In some embodiments, the promoter in the expression cassette is a target cell selective promoter. In some embodiments, the target cell selective promoter is selected from one of the following: a cardiomyocyte selective promoter, a nucleus pulposus cell selective promoter, a kidney cell selective promoter, a hepatocyte selective promoter, an endothelial progenitor cell selective promoter, a chondrocyte selective promoter, a glial cell selective promoter, a neural progenitor cell selective promoter, and a neuron selective promoter. Suitable cardiomyocyte selective promoters include, but are not limited to, a cardiac-specific troponin T promoter (TNNT2), an atrial natriuretic factor (ANF) promoter, and a Na + -Ca + Exchange body (NCX1) promoter. An exemplary nucleus pulposus selective promoter is carbonic anhydrase 12 (CA12) promoter. Exemplary kidney selective promoters include kidney-specific cadherin (KSPC) Na / glucose cotransporter (SGLT2) gene promoters in the proximal tubule, and 2 chloride ion cotransporter (NKCC2) gene promoters for expression in the thick ascending limb of Henle's loop. An exemplary chondrocyte selective promoter is type II collagen (COL2A1) promoter. Exemplary liver selective promoters are serum albumin gene promoter and alpha-1-antitrypsin gene promoter. Exemplary glial selective promoters include glial fibrillary acid protein GFAP gene promoter of astrocytes and S100β gene promoter of astrocytes and myelin oligodendrocytes. Exemplary neuron selective promoters include: CamKII gene promoter and synapsin gene promoter.

[0097] In some embodiments, where the gRNA is to be expressed as part of a targeted transactivator, a suitable promoter for expressing the gRNA is a PolIII promoter, such as a U3 or U6 promoter. In other embodiments, the expression of both the gRNA and the targeting enzyme (e.g., dCas9) (i.e., both components of the targeted transactivator) can be driven by a single promoter (e.g., H1 promoter), which can be utilized by both RNA polymerases II and III (Gao et al., 2018, Mol. Ther. Nucleic Acids, 14: 32-40).

[0098] In some embodiments, the promoter in the expression cassette is an inducible (and reversible) promoter, referred to as an "inducible promoter". Typically, an inducible promoter as mentioned herein is a promoter whose activity is controlled by an exogenously provided inducible transactivator, which can be conditionally activated or inactivated. In some embodiments, the transactivator is a ligand-regulated or light-regulated transactivator that can transactivate an inducible promoter under expression-permitting conditions. Suitable inducible transactivators include, but are not limited to, reverse tetracycline transactivators (rtTA), tetracycline (off) transactivators (tTa), cumate-inducible transactivators, FK506-inducible transactivators, and red / far-red light-inducible REDMAP transactivators (see, for example, Zhou et al., 2022, Nature Biotechnology, 40 (2): 262-272). Inducible promoters and transactivation systems are particularly useful in the context of partial reprogramming because their use allows for fine control over the timing of expression of reprogramming factors in a subject treated according to the methods disclosed herein.

[0099] In some embodiments, at least one of the exogenous nucleic acids provided to the subject to be treated is a polycistronic, that is, it encodes two or more proteins (e.g., reprogramming factors). In some embodiments, polycistronic synthetic mRNA is provided. In other embodiments, the nucleic acid provided comprises a polycistronic expression cassette ("polycistronic expression cassette") encoding a polycistronic mRNA, and the mRNA produces an independent polypeptide comprising different amino acid sequences or functions after translation. In some embodiments, the polycistronic expression cassette encodes a "polyprotein", which comprises a plurality of polypeptide sequences separated by picornavirus encoding, such as foot-and-mouth disease virus (foot-and-mouth diseasevirus, FMDV) virus 2A peptide sequence. The 2A peptide sequence plays a synergistic translation role by preventing the formation of a normal peptide bond between the conservative glycine and the last proline, thereby causing the ribosome to jump to the next codon and the nascent peptide cutting between Gly and Pro. After cleavage, the short 2A peptide remains fused to the C-terminus of the "upstream" protein, while a proline is added to the N-terminus of the "downstream" protein, which allows the nascent polypeptide sequence to be cleaved into separate polypeptides during translation (see, e.g., Trichas et al., (2008), BMC Biology 6:40).

[0100] In other embodiments, the polycistronic mRNA or expression cassette incorporates one or more internal ribosome entry site (IRES) sequences. IRES sequences and their uses are known in the art, as exemplified in, for example, Martinez-Sales, (1999), Current Opinion in Biotechnology, 10: 458-464.

[0101] In some embodiments, one or more exogenous nucleic acids use a combination of 2A and IRES elements in continuous nucleic acids. In some embodiments, a single expression cassette or synthetic mRNA encodes four reprogramming factors (RFs) in the following configuration: RF1-2a-RF2-IRES-RF3-2a-RF4. This configuration is illustrated in, for example, Lehmann et al., (2019), Current Gene Therapy, 19 (4): 248-254, where a polycistronic expression cassette OCT4-2a-KLF4-IRES-SOX2-2a-c-MYC is used for partial reprogramming. In other embodiments, two separate expression cassettes are provided as RF1-2a-RF2 and RF3-2a-RF4.

[0102] Therefore, the present invention also provides a method for enhancing partial reprogramming of target cells in a postnatal subject in need thereof, the method comprising administering to the subject a plurality of mesenchymal lineage progenitor or stem cells (MLPSCs), exosomes derived therefrom, or conditioned medium derived from a culture of the plurality of MLPSCs, wherein the subject expresses exogenous reprogramming factors comprising OCT4, SOX2, KLF4 and c-MYC in a target cell population simultaneously with or after administration, wherein one or more exogenous nucleic acids for expressing the exogenous reprogramming factors have been delivered to the target cell population (i) as one or more synthetic mRNAs; (ii) by one or more non-integrating recombinant viruses; or (iii) as a combination of (i) and (ii); thereby a plurality of the target cells in the postnatal subject are partially reprogrammed.

[0103] Also provided is a composition for enhancing partial reprogramming of target cells, comprising a plurality of modified mesenchymal lineage progenitor or stem cells (MLPSCs), the MLPSCs comprising one or more exogenous nucleic acids encoding one or more of: (a) reprogramming factors; or (b) targeted transactivators that induce expression of one or more endogenous reprogramming factors, wherein: (i) when one or more target cells are present, the modified MLPSCs deliver the one or more exogenous nucleic acids to the one or more target cells; (ii) the one or more encoded reprogramming factors are expressed in the one or more target cells at a level sufficient to partially reprogram the one or more target cells; and (iii) the one or more encoded reprogramming factors or targeted transactivators are substantially incapable of being expressed in the modified MLPSCs prior to the delivery in (i).

[0104] Also disclosed herein is a composition for enhancing partial reprogramming of target cells, comprising:

[0105] (i) culture amplified human mesenchymal stem cell (hMSC) population; (ii) conditional replication helper virus; and (iii) one or more helper-dependent viruses, the viruses: (a) comprising one or more reprogramming factors or one or more expression cassettes of targeted transactivators that induce the expression of one or more endogenous reprogramming factors; and (b) substantially free of viral coding sequences. In some preferred embodiments, the conditional replication helper virus and the helper-dependent reprogramming virus are adenoviruses. It is known that conditional replication helper adenoviruses and helper-dependent adenoviruses replicate at sufficient levels in hMSCs to amplify and secrete, thereby spreading to nearby cells. See McKenna et al., 2021, Mol. Therapy, 29 (5): 1808-1820. In some embodiments, helper adenoviruses and helper-dependent adenoviruses are both serotype 5 adenoviruses. It is believed that the use of hMSCs for reprogramming viruses can protect such viruses from immune neutralization during partial reprogramming in vivo. In some embodiments, such compositions also include one or more of prostaglandin E2 or blocking antibodies against natural killer cell receptor NKG2D. In some embodiments, the aforementioned compositions are used in methods for enhancing partial reprogramming in vivo.

[0106] In the preferred embodiment of the partial reprogramming method disclosed herein, one or more reprogramming factors as disclosed herein can be inducibly and reversibly expressed for at least two consecutive days but no more than about a period of time of 21 consecutive days, such as 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 14 days, 15 days, 16 days, 18 days, 20 days, or at least two consecutive days to another consecutive number of days of 21 consecutive days. The expression period of limiting the reprogramming factor in the target cell group reduces the possibility of developing from partial reprogramming to complete reprogramming and losing target cell identity, which is undesirable. In some preferred embodiments, one or more reprogramming factors can be inducibly and reversibly expressed for at least two consecutive days but no more than 15 days.

[0107] In some embodiments, one or more reprogramming factors are expressed for at least two days but not longer than a cumulative time period of about 21 days, such as 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 14 days, 15 days, 16 days, 18 days, 20 days, or at least two days but no more than another cumulative time period of 21 days. For example, one or more reprogramming factors can be expressed for seven days in the first time period, then hardly expressed or not expressed for about seven days, then one or more reprogramming factors are expressed for about seven days in the second time period, and then the expression of one or more reprogramming factors is terminated.

[0108] Mesenchymal lineage precursors or stem cells

[0109] As used herein, the term "mesenchymal lineage precursor or stem cell (MLPSC)" refers to an undifferentiated pluripotent cell that has the ability to self-renew while maintaining pluripotency and the ability to differentiate into cell types of many mesenchymal origins (e.g., osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts, and tendons) or non-mesenchymal origins (e.g., hepatocytes, neural cells, and epithelial cells). For the avoidance of doubt, "mesenchymal lineage precursor cells" refer to cells that can differentiate into mesenchymal cells (such as bone, cartilage, muscle, and adipocytes) as well as fibrous connective tissue.

[0110] The term "mesenchymal lineage precursor or stem cell" includes both parental cells and their undifferentiated offspring. The term also includes mesenchymal precursor cells, multipotent stromal cells, mesenchymal stem cells (MSC), perivascular mesenchymal precursor cells and their undifferentiated offspring. Mesenchymal lineage precursors or stem cells can be autologous, allogeneic, xenogeneic, homologous or isogenic. Autologous cells are separated from the same individual to which they will be reimplanted. Allogeneic cells are separated from donors of the same species. Xenogeneic cells are separated from donors of another species. Homologous or isogenic cells are separated from genetically identical organisms, such as twins, clones, or highly inbred research animal models.

[0111] In one example, the mesenchymal lineage precursors or stem cells are allogeneic. In one example, the allogeneic mesenchymal lineage precursors or stem cells are culture expanded and cryopreserved.

[0112] Mesenchymal lineage precursors or stem cells mainly reside in the bone marrow, but have also been shown to be present in a variety of host tissues, including, for example, umbilical cord blood and umbilical cord, adult peripheral blood, adipose tissue, trabecular bone and dental pulp. They are also present in skin, spleen, pancreas, brain, kidney, liver, heart, retina, brain, hair follicles, intestines, lungs, lymph nodes, thymus, ligaments, tendons, skeletal muscle, dermis and periosteum; and can be differentiated into reproductive system such as mesoderm and / or endoderm and / or ectoderm. Therefore, mesenchymal lineage precursors or stem cells can be differentiated into a large number of cell types, including but not limited to adipose tissue, bone tissue, cartilage tissue, elastic tissue, muscle tissue and fibrous connective tissue. The specific lineage-stereotype and differentiation pathways that these cells enter depend on the various effects from mechanical influences and / or endogenous bioactive factors (such as growth factors, cytokines and / or local microenvironment conditions established by host tissues).

[0113] The term "enriched", "enriched" or its variants are used herein to describe a cell population, wherein the ratio of a particular cell type or the ratio of many particular cell types is increased when compared to an untreated cell population (e.g., a cell in its native environment). In one example, the group enriched for mesenchymal lineage precursors or stem cells comprises at least about 0.1%, or 0.5%, or 1%, or 2%, or 5%, or 10%, or 15%, or 20%, or 25%, or 30%, or 50%, or 75% of mesenchymal lineage precursors or stem cells. In this regard, the term "enriched mesenchymal lineage precursors or stem cell cell population" will be understood as the term "comprising X% mesenchymal lineage precursors or stem cell cell populations" provides clear support, wherein X% is a percentage as listed herein. In some examples, mesenchymal lineage precursor cells or stem cells can form clonogenic colonies, such as CFU-F (fibroblasts) or a subset thereof (eg, 50% or 60% or 70% or 70% or 90% or 95%) can have this activity.

[0114] In one example of the present disclosure, the mesenchymal lineage precursor or stem cell is a mesenchymal stem cell (MSC). MSC can be a homogenous composition or can be a mixed cell population enriched with MSC. Homogeneous MSC compositions can be obtained by culturing adherent bone marrow or periosteal cells, and MSC can be identified by specific cell surface markers identified with unique monoclonal antibodies. The method for obtaining a cell population enriched with MSC is described in, for example, U.S. Patent No. 5,486,359. Alternative sources of MSC include, but are not limited to, blood, skin, cord blood, muscle, fat, bone, and perichondrium. In one example, MSC is allogenic. In one example, MSC is cryopreserved. In one example, MSC is cultured, amplified, and cryopreserved.

[0115] In another example, the mesenchymal lineage precursor or stem cell is a CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, MHC1+ MSC.

[0116] In one example, mesenchymal lineage precursors or stem cells are cultured and expanded from MSC groups expressing markers (including CD73, CD90, CD105 and CD166) and lacking hematopoietic cell surface antigens (such as CD45 and CD31). For example, mesenchymal lineage precursors or stem cells can be cultured and expanded from MSC groups of CD73+, CD90+, CD105+, CD166+, CD45- and CD31-. In one example, the further feature of the MSC group is that the level of class I major histocompatibility complex (MHC) is low. In another example, MSC is negative for class II major histocompatibility complex molecules, and negative for costimulatory molecules CD40, CD80 and CD86. In one example, culture expansion includes 5 passages.

[0117] The isolated or enriched mesenchymal lineage precursors or stem cells can be expanded in vitro by culture.The isolated or enriched mesenchymal lineage precursors or stem cells can be cryopreserved, thawed and subsequently expanded in vitro by culture.

[0118] In one example, the isolated or enriched mesenchymal lineage precursors or stem cells are cultured at 50,000 viable cells / cm 2 The cells are plated in culture medium (serum-free or serum-supplemented), for example, alpha minimum essential medium (αMEM) supplemented with 5% fetal bovine serum (FBS) and glutamine, and allowed to adhere to the culture vessels overnight at 37° C., 20% O 2. The culture medium is then replaced and / or changed as needed, and the cells are cultured at 37° C., 5% O 2 for an additional 68 to 72 hours.

[0119] As will be appreciated by those skilled in the art, the mesenchymal lineage precursors or stem cells cultured are phenotypically different from cells in vivo. For example, in one embodiment, they express one or more of the following markers: CD44, NG2, DC146, and CD140b. Cultivated mesenchymal lineage precursors or stem cells are also biologically different from cells in vivo, having a higher proliferation rate than most non-circulating (quiescent) cells in vivo.

[0120] In one example, a cell population is enriched from a cell preparation comprising STRO-1+ cells in a selectable form. In this regard, the term "selectable form" is understood to mean that the cell expresses a marker (e.g., a cell surface marker) that allows the selection of STRO-1+ cells. The marker may be STRO-1, but not necessarily. For example, as described herein and / or illustrated, cells (e.g., mesenchymal precursor cells) expressing STRO-2 and / or STRO-3 (TNAP) and / or STRO-4 and / or VCAM-1 and / or CD146 and / or 3G5 also express STRO-1 (and may be STRO-1 bright). Therefore, the indication that the cell is STRO-1+ does not mean that the cell is selected only by STRO-1 expression. In one example, cells are selected based at least on STRO-3 expression, for example, they are STRO-3+ (TNAP+).

[0121] Reference to the selection of cells or groups thereof does not necessarily require selection from a specific tissue source. As described herein, STRO-1+ cells can be selected from or isolated from or enriched from a variety of sources. That is, in some instances, these terms provide support for selection from any tissue or vascularized tissue containing STRO-1+ cells (e.g., mesenchymal precursor cells) or tissue containing pericytes (e.g., STRO-1+ pericytes) or any one or more of the tissues listed herein.

[0122] In one example, the cells used in the present disclosure express one or more markers, alone or collectively, selected from the group consisting of TNAP+, VCAM-1+, THY-1+, STRO-2+, STRO-4+ (HSP-90β), CD45+, CD146+, 3G5+, or any combination thereof.

[0123] "Individually" means that the disclosure individually encompasses the recited marker or marker groups, and although individual markers or marker groups may not be individually listed herein, the appended claims may define such markers or marker groups individually and separately from each other.

[0124] "Collectively" means that the present disclosure encompasses any number or combination of the listed markers or marker groups, and although such number or combination of markers or marker groups may not be specifically listed herein, the appended claims may define such combinations or sub-combinations individually and separately from any other combination of markers or marker groups.

[0125] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue nonspecific alkaline phosphatase. For example, the term encompasses liver isoforms (LAP), bone isoforms (BAP), and kidney isoforms (KAP). In one example, TNAP is BAP. In one example, TNAP as used herein refers to a molecule that can bind to the STRO-3 antibody produced by a hybridoma cell line deposited with the ATCC on December 19, 2005 under the provisions of the Budapest Treaty under deposit accession number PTA-7282.

[0126] Furthermore, in one example, STRO-1+ cells were able to generate clonogenic CFU-F.

[0127] In one example, a significant proportion of STRO-1+ cells are able to differentiate into at least two different germ lineages. Non-limiting examples of lineages to which STRO-1+ cells can be committed include bone precursor cells; hepatocyte progenitor cells, which are multipotent for bile duct epithelial cells and hepatocytes; neural restricted cells, which can generate glial precursors that progress to oligodendrocytes and astrocytes; neuronal precursors that progress to neurons; myocardium and precursors of cardiomyocytes; pancreatic beta cell lineage that secretes glucose-responsive insulin. Other lineages include, but are not limited to, odontoblasts, dentin-producing cells, and chondrocytes, as well as precursors of the following cells: retinal pigment epithelial cells, fibroblasts, skin cells (such as keratinocytes), dendritic cells, hair follicle cells, renal ductal epithelial cells, smooth and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiomyocytes, smooth muscle cells, skeletal muscle cells, pericytes, vascular cells, epithelial cells, glial cells, neurons, astrocytes, and oligodendrocytes.

[0128] In one example, mesenchymal lineage precursors or stem cells are obtained from a single donor or multiple donors, wherein the donor samples or mesenchymal lineage precursors or stem cells are subsequently pooled and then expanded in culture.

[0129] MLPSCs encompassed by the present disclosure may also be cryopreserved prior to administration to a subject. In one example, mesenchymal lineage precursors or stem cells are culture expanded and cryopreserved prior to administration to a subject.

[0130] In one example, the present disclosure encompasses mesenchymal lineage precursors or stem cells and their progeny, soluble factors derived therefrom and / or extracellular vesicles isolated therefrom. In another example, the present disclosure encompasses mesenchymal lineage precursors or stem cells and extracellular vesicles isolated therefrom. For example, the mesenchymal lineage precursors or stem cells of the present disclosure can be cultured and expanded for a period of time under conditions suitable for secretion of extracellular vesicles into cell culture medium. Secreted extracellular vesicles can then be obtained from the culture medium for therapy.

[0131] As used herein, the term "extracellular vesicle" refers to lipid particles that are naturally released from cells and range in size from about 30 nm to as large as 10 microns, but the size of the lipid particles is usually less than 200 nm. They may contain extracellular matter from releasing cells (e.g., mesenchymal stem cells; STRO-1 + cells) proteins, nucleic acids, lipids, metabolites or organelles.

[0132] As used herein, the term "exosome" refers to a type of extracellular vesicle that is generally in the range of about 30 nm to about 150 nm in size and originates from the endosomal compartment of mammalian cells, from which it is transported to the cell membrane and released. They may contain nucleic acids (e.g., RNA; microRNA), proteins, lipids, and metabolites, and play a role in intercellular communication by being secreted from one cell and taken up by other cells to deliver their cargo.

[0133] MLPSC can be found in bone marrow, blood, dental pulp cells, adipose tissue, skin, spleen, pancreas, brain, kidney, liver, heart, retina, brain, hair follicles, intestines, lungs, lymph nodes, thymus, bones, ligaments, tendons, skeletal muscle, dermis and periosteum; and can differentiate into germ lines such as mesoderm and / or endoderm and / or ectoderm. In some preferred embodiments, MLPSC is from bone marrow, dental pulp or adipose tissue, more preferably from dental pulp or adipose tissue. Therefore, MLPSC can differentiate into a large number of cell types, including but not limited to adipose tissue, bone tissue, cartilage tissue, elastic tissue, muscle tissue and fibrous connective tissue. The specific lineage-stereotype and differentiation pathways that these cells enter depend on various effects from mechanical influences and / or endogenous bioactive factors (such as growth factors, cytokines and / or local microenvironment conditions established by host tissues). Therefore, MLPSC cells are non-hematopoietic progenitor cells, and the daughter cells produced by their division are stem cells or precursor cells, which will eventually be irreversibly differentiated to produce phenotypic cells.

[0134] In a preferred embodiment, the MLPSC cells used in the methods disclosed herein are enriched from a sample obtained from a subject (e.g., a subject to be treated or a related subject or an unrelated subject (whether the same species or a different species)). This enrichment can be performed ex vivo or in vitro. The terms "enriched", "enriched" or variations thereof are used herein to describe a cell population in which the proportion of a particular cell type or the proportion of a number of particular cell types is increased when compared to an untreated population.

[0135] In some preferred embodiments, the cells used in the present invention are individually or collectively positive for the expression of more markers selected from the group consisting of: STRO-1 + TNAP + 、VCAM-1 + 、THY-1 + 、STRO-2 + 、CD146 + 、3G5 + Or any combination thereof. In some preferred embodiments, MLPSCs are enriched for STRO-1 + Mesenchymal precursor cells (MPCs) or their culture-expanded multipotent progeny.

[0136] Preferably, STRO-1 + STRO-1 亮 (Synonymous STRO-1 bri ). Preferably, STRO-1 亮 TNAP + 、VCAM-1 + 、THY-1 + 、STRO-2 + and / or CD146 + In one embodiment, the MLPSC is a perivascular mesenchymal precursor cell as defined in WO 2004 / 85630.

[0137] In some embodiments, the culture-expanded MLPSCs express one or more markers collectively or individually selected from the group consisting of TNF-R1, TGFβ1, LFA-3, THY-1, VCAM-1, ICAM-1, PECAM-1, P-selectin, L-selectin, 3G5, CD49a / CD49b / CD29, CD49c / CD29, CD49d / CD29, CD 90, CD29, CD18, CD61, integrin β6-19, thrombomodulin, CD10, CD13, SCF, PDGF-R, EGF-R, IGF1-R, NGF-R, FGF-R, leptin-R (STRO-2=leptin-R), RANKL, STRO-1, CD146, or any combination of these markers.

[0138] Cells referred to as "positive" for a given marker may express low (lo or dim) or high (bright, bri) levels of the marker, depending on the extent to which the marker is present on the cell surface, where the term is related to the fluorescence intensity or other markers used in the cell sorting process. The difference between lo (or dim or dull) and bri can be understood in the context of the marker used when sorting a particular cell population. Cells referred to as "negative" for a given marker are not necessarily completely absent from the cell. This term means that the cell expresses the marker at a relatively very low level, and the marker produces a very low signal when detectably labeled or is undetectable above background levels.

[0139] As used herein, the term "bright" refers to a cell surface marker that produces a relatively high signal when detectably labeled. While not wishing to be bound by theory, it is proposed that "bright" cells express more target marker protein (e.g., an antigen recognized by STRO-1) than other cells in the sample. For example, as determined by fluorescence activated cell sorting (FACS) analysis, when labeled with a FITC-conjugated STRO-1 antibody, STRO-1 bri cells than non-bright cells (STRO-1 dull / dim ) produces a stronger fluorescent signal. Preferably, the "bright" cells constitute at least about 0.1% of the brightest labeled bone marrow mononuclear cells contained in the starting sample. In other embodiments, the "bright" cells constitute at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, or at least about 2% of the brightest labeled bone marrow mononuclear cells contained in the starting sample. In a preferred embodiment, the "bright" cells constitute at least about 0.1%, at least about 0.5%, at least about 1%, at least about 1.5%, or at least about 2% of the brightest labeled bone marrow mononuclear cells contained in the starting sample. - cells), STRO-1 亮 cells had a 2-log higher expression of STRO-1 on the surface. dimand / or STRO-1 中间 The cells have surface expression of STRO-1 that is less than 2 logs above "background," typically about 1 log or less.

[0140] As used herein, the term "TNAP" is intended to encompass all isoforms of tissue nonspecific alkaline phosphatase. For example, the term encompasses liver isoforms (LAP), bone isoforms (BAP), and kidney isoforms (KAP). In a preferred embodiment, TNAP is BAP. In a particularly preferred embodiment, TNAP as used herein refers to a molecule that binds to a STRO-3 antibody produced by a hybridoma cell line deposited with the ATCC on December 19, 2005 under the provisions of the Budapest Treaty, with accession number PTA-7282.

[0141] In addition, in a preferred embodiment, STRO-1 + Pluripotent cells are able to generate clonogenic CFU-F.

[0142] Preferably, a significant proportion of MLPSCs are capable of differentiating into at least two different germ lineages. Non-limiting examples of lineages to which multipotent cells can be committed include bone precursor cells; hepatocyte progenitor cells, which are multipotent for bile duct epithelial cells and hepatocytes; neural restricted cells, which can generate glial cell precursors that progress to oligodendrocytes and astrocytes; neuronal precursors that progress to neurons; myocardium and precursors of myocardial cells; pancreatic beta cell lineages that secrete glucose-responsive insulin. Other lineages include, but are not limited to, odontoblasts, dentin-producing cells, and chondrocytes, as well as precursors of the following cells: retinal pigment epithelial cells, fibroblasts, skin cells (such as keratinocytes), dendritic cells, hair follicle cells, renal ductal epithelial cells, smooth and skeletal muscle cells, testicular progenitor cells, vascular endothelial cells, tendons, ligaments, cartilage, adipocytes, fibroblasts, bone marrow stroma, cardiomyocytes, smooth muscle cells, skeletal muscle cells, pericytes, vascular cells, epithelial cells, glial cells, neurons, astrocytes, and oligodendrocytes.

[0143] In another embodiment, the MLPSCs are unable to generate hematopoietic cells upon culture.

[0144] In one embodiment, MLPSCs are harvested from a subject to be treated and cultured in vitro using standard techniques, e.g., prior to use in a therapeutic method as described herein according to any embodiment.

[0145] Culture-expanded MLPSCs can be obtained by culturing in any suitable culture medium known in the art, for example, as described in WO 2004 / 85630. In one embodiment, the progeny cells used in the method of the present invention are isolated from bone marrow using magnetic beads labeled with STRO-3 antibodies. + STRO-1 + pluripotent cells, and then culturing and expanding the isolated cells (see Gronthos et al., Blood 85:929-940, 1995 for examples of suitable culture conditions).

[0146] It should be understood that in carrying out the present invention, the isolation of cells carrying any given cell surface marker can be achieved by many different methods, however, the preferred method relies on binding a binding agent (e.g., an antibody or antigen binding fragment thereof) to the marker of interest, followed by separation of cells that exhibit binding (high level binding, low level binding or no binding). The most convenient binding agent is an antibody or antibody-based molecule, preferably a monoclonal antibody or monoclonal antibody-based molecule, because these latter agents have specificity. Antibodies can be used in both steps, however other agents can also be used, so ligands for these markers can also be used to enrich for cells carrying them or lacking them.

[0147] The antibody or ligand can be attached to a solid support to allow for rough separation. Separation techniques preferably increase the retention of the vigor of the part to be collected to the greatest extent. Relatively rough separation can be obtained using various techniques of different efficacy. The specific technique employed will depend on separation efficiency, related cytotoxicity, the ease and speed of carrying out, and the need for complex equipment and / or technical skills. Separation procedures may include but are not limited to magnetic separation, use antibody-coated magnetic beads, affinity chromatography, and use antibodies attached to a solid matrix to carry out "panning". The technology providing accurate separation includes but is not limited to FACS. The method for carrying out FACS will be apparent to those skilled in the art.

[0148] Antibodies to each of the markers described herein are commercially available (eg, monoclonal antibodies to STRO-1 or TNAP are commercially available from R&D Systems, USA), may be obtained from the ATCC or other depository organizations, and / or may be generated using art-recognized techniques.

[0149] Preferably, the method for isolating MLPSCs comprises, for example, a first step, a solid phase sorting step, using, for example, magnetic activated cell sorting (MACS) that identifies high levels of STRO-1 expression. If desired, a second sorting step may then be performed to produce higher levels of precursor cell expression as described in patent specification WO 01 / 14268. This second sorting step may involve the use of two or more markers.

[0150] The method of obtaining MLPSC cells may also include harvesting the cell source using known techniques before the first enrichment step. Therefore, the tissue will be surgically removed. Then, the cells constituting the source tissue will be separated into a so-called single cell suspension. This separation can be achieved by physical or enzymatic means.

[0151] In some embodiments, the MLPSCs (including culture-expanded MLPSCs) have been cryopreserved and thawed.In the most preferred embodiments, the MLPSCs used in the treatment methods disclosed herein are human MLPSCs.

[0152] Culture and expansion of MLPSCs

[0153] In one example, MLPSC is culture-expanded. The difference between "culture-expanded" mesenchymal lineage precursors or stem cell culture media and freshly isolated cells is that they have been cultured and passaged (i.e., passage culture) in cell culture media. In one example, culture-expanded mesenchymal lineage precursors or stem cells are cultured and expanded for about 4-10 passages. In some embodiments, culture-expanded MLPSC is pre-licensed by exposure to certain proinflammatory cytokines as disclosed herein. In one example, mesenchymal lineage precursors or stem cells are cultured and expanded for at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 passages. For example, mesenchymal lineage precursors or stem cells can be cultured and expanded for at least 5 passages. In one example, mesenchymal lineage precursors or stem cells can be cultured and expanded for at least 5-10 passages. In one example, mesenchymal lineage precursors or stem cells can be cultured and expanded for at least 5-8 passages. In one example, mesenchymal lineage precursors or stem cells can be cultured and expanded for at least 5-7 passages. In one example, mesenchymal lineage precursors or stem cells can be cultured and expanded for more than 10 passages. In another example, mesenchymal lineage precursors or stem cells can be cultured and expanded for more than 7 passages. In these examples, stem cells can be cultured and expanded before being cryopreserved to provide an intermediate cryopreserved MLPSC group. In one example, the composition of the present disclosure is prepared from an intermediate cryopreserved MLPSC group. For example, the intermediate cryopreserved MLPSC group can be further cultured and expanded before administration, as further discussed below. Therefore, in one example, mesenchymal lineage precursors or stem cells are cultured and expanded and cryopreserved. In one embodiment of these examples, mesenchymal lineage precursors or stem cells can be obtained from a single donor or multiple donors, wherein donor samples or mesenchymal lineage precursors or stem cells are subsequently collected and then cultured and expanded. In one example, the culture expansion process includes:

[0154] -i. expanding the number of viable cells by passage expansion to provide a preparation of at least about 1 billion viable cells, wherein the passage expansion comprises establishing a primary culture of the isolated mesenchymal lineage precursors or stem cells and then successively establishing a first non-primary (P1) culture of the isolated mesenchymal lineage precursors or stem cells from the previous culture;

[0155] -ii. expanding the P1 culture of the isolated mesenchymal lineage precursors or stem cells into a second non-primary (P2) culture of the mesenchymal lineage precursors or stem cells by subculture expansion; and,

[0156] -iii. preparing and cryopreserving an intermediate mesenchymal lineage precursor or stem cell preparation obtained from a P2 culture of mesenchymal lineage precursors or stem cells; and,

[0157] -iv. thawing the cryopreserved in-process intermediate mesenchymal lineage precursor or stem cell preparation and expanding the in-process intermediate mesenchymal lineage precursor or stem cell preparation by passage expansion.

[0158] In one example, the culture-expanded mesenchymal lineage precursors or stem cells are culture-expanded for about 4-10 passages, wherein the mesenchymal lineage precursors or stem cells are cryopreserved at least 2 passages or 3 passages before further culture expansion. In one example, the mesenchymal lineage precursors or stem cells are culture-expanded for at least 1, at least 2, at least 3, at least 4, at least 5 passages, cryopreserved, and then further culture-expanded for at least 1, at least 2, at least 3, at least 4, at least 5 passages, and then administered or further cryopreserved.

[0159] In one example, mesenchymal lineage precursors or stem cells are expanded in culture in a cell factory, such as a 5-layer or 10-layer cell factory.

[0160] In one example, most of the mesenchymal lineage precursors or stem cells in the compositions of the present disclosure have about the same number of generations (i.e., they are within about 1, or about 2, or about 3, or about 4 cell doublings of each other). In one example, the average number of cell doublings in the compositions of the present invention is about 20 to about 25 doublings. In one example, the average number of cell doublings in the compositions of the present invention is about 9 to about 13 (e.g., about 11 or about 11.2) doublings generated from the primary culture, plus about 1, about 2, about 3, or about 4 doublings per passage (e.g., about 2.5 doublings per passage). Exemplary average cell doublings in the compositions of the invention are any of about 13.5, about 16, about 18.5, about 21, about 23.5, about 26, about 28.5, about 31, about 33.5, and about 36 when generated through about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, and about 10 passages, respectively.

[0161] Any equipment and cell processing methods known in the art can be used to separate and amplify mesenchymal lineage precursors or stem cells in vitro. Various culture expansion embodiments disclosed herein use steps that require manipulation of cells, such as inoculation, feeding, dissociation of adherent cultures or washing steps. Any step of manipulating cells is likely to damage the cells. Although mesenchymal lineage precursors or stem cells can generally tolerate a certain amount of damage during preparation, it is preferred to manipulate cells by fully performing one or more given steps of the operating procedure and / or equipment while minimizing damage to the cells.

[0162] In one example, mesenchymal lineage precursors or stem cells are washed in an apparatus comprising a cell source bag, a wash solution bag, a recirculating wash bag, a rotating membrane filter having an inlet port and an outlet port, a filtrate bag, a mixing zone, a final product bag for the washed cells, and appropriate tubing, e.g., as described in US 6,251,295, which is hereby incorporated by reference.

[0163] In one example, in terms of being CD105 positive and CD166 positive and CD45 negative, the mesenchymal lineage precursor or stem cell composition according to the present disclosure is 95% homogeneous. In one example, this homogeneity persists by ex vivo amplification (i.e., by multiple population doublings). In one example, the composition comprises at least one therapeutic dose of mesenchymal lineage precursors or stem cells, and the mesenchymal lineage precursors or stem cells comprise less than about 1.25% of CD45+ cells, at least about 95% of CD105+ cells, and at least about 95% of CD166+ cells. In one example, this homogeneity persists after cryogenic storage and thawing, wherein the cells generally also have about 70% or higher viability.

[0164] Modified MLPSCs and related uses

[0165] In some embodiments, the methods disclosed herein utilize modified (e.g., genetically modified MLPSC). Although not wishing to be bound by theory, it is known that MLPSC (e.g., hMSC) can deliver nucleic acids (e.g., miRNA, antagomiR, siRNA) to other cells (e.g., target cells) via two gap junctions, exosomes, and extracellular vesicles. In some embodiments, the modified MLPSC comprises at least one of exogenous anti-inflammatory miRNA, antagomiR, siRNA, RNAi, antisense oligonucleotides, or antisense RNA, which can be used to further promote partial reprogramming of target cells. In some embodiments, the modified MLPSC comprises one or more exogenous anti-inflammatory miRNAs selected from the group consisting of: miR-10a, miR-21, miR-24, miR-124, miR-145, miR-146, miR-149, and miR-181a-miR-181d. In other embodiments, the modified MLPSCs comprise one or more exogenous anti-inflammatory antagomiRs selected from the group consisting of miR-146b-5p, miR-34a antagomir, miR-21 antagomir, miR-126 antagomir, miR-221 antagomir, miR-338-3pan tagomir, and miR-155 antagomir. In some embodiments, the modified MLPSCs comprise one or more exogenous anti-inflammatory siRNAs, antisense oligonucleotides, or antisense RNAs targeting one or more targets selected from the group consisting of TNF-α, IL-1β, and IL-18.

[0166] In some embodiments, the modified MLPSCs comprise a gene targeting p16 INK4a 、p21 WAF1 / CIP1 In some preferred embodiments, the modified MLPSCs comprise at least one of an exogenous siRNA, RNAi, antisense oligonucleotide, or antisense RNA targeting at least one of the mTOR signaling pathway or the c-Jun N-terminal kinase (JNK) signaling pathway. INK4a 、p21 WAF1 / CIP1 or exogenous siRNA or antisense oligonucleotides to one or more of mTOR.

[0167] In some embodiments, the modified MLPSc comprises a miRNA selected from the group consisting of mir-302b, mir-302c, mir-302a, mir-302d, and mir-367, which can increase the efficiency of partial reprogramming.

[0168] In some embodiments, the modified MLPSC is a genetically modified MLPSC comprising one or more exogenous nucleic acids encoding one or more reprogramming factors or one or more targeted transactivators, wherein:

[0169] (i) when one or more target cells are present, the modified MLPSC delivers one or more exogenous nucleic acids to the one or more target cells;

[0170] (ii) one or more encoded reprogramming factors are expressed in the one or more target cells at a level sufficient to partially reprogram the one or more target cells; and

[0171] (iii) one or more encoded reprogramming factors or targeted transactivators are not substantially expressed in the modified MLPSC before delivery in (i) to avoid reprogramming of the modified MLPSC itself. Although it is not desired to be bound by theory, it is generally understood in the art that mRNA can be delivered between cells via tunnel nanotubes. See, for example, Haimovich et al. (2021), Biochem Soc Trans., 49 (1): 145-160. Therefore, in some preferred embodiments, the genetically modified MLPSC is loaded with one or more synthetic mRNAs, which encode one or more reprogramming factors or targeted transactivators as described herein, wherein the encoded reprogramming factor is a fusion protein comprising a CDD as described herein, which prevents the accumulation of encoded proteins in the absence of a stabilizing ligand (e.g., trimethoprim). This configuration allows the delivery of synthetic mRNA to target cells.

[0172] The efficiency of genetic modification can rarely reach 100%, and it is usually desirable to enrich the cell group that has been successfully modified. In one example, the modified cells can be enriched by utilizing the functional characteristics of the new genotype. An exemplary method for enriching modified cells is the positive selection using selectable or screenable marker genes. "Marker gene" refers to a gene that gives a unique phenotype to the cells expressing the marker gene and therefore allows such transformed cells to be distinguished from cells without markers. Selectable marker genes give people the characteristic of "selection" based on resistance to selection agents (e.g., antibiotics such as puromycin (puromycin) or blasticidin (blasticidin)). Screenable marker genes (or reporter genes) give people the characteristic (e.g., β-glucuronidase, luciferase, GFP or other enzyme activities not present in untransformed cells) that can be identified by observation or testing (i.e., by "screening"). In one example, genetically modified mesenchymal lineage precursors or stem cells are selected based on resistance to drugs (such as neomycin (neomycin)) or colorimetric selection based on lacZ expression.

[0173] In some embodiments, the genetically modified MLPSCs described above are provided as a composition for enhancing partial reprogramming of target cells, which also serves as a delivery platform (e.g., a combined helper / helper-dependent viral platform; Fig. 9 ) to deliver exogenous nucleic acids encoding one or more of the following to the target cells of the subject: (a) reprogramming factors; or (b) targeted transactivators that induce the expression of one or more endogenous reprogramming factors. As described above, by judiciously selecting encoding nucleic acids, nucleic acid transfer from genetically modified MLPSC occurs before expressing reprogramming factors in target cells at functionally relevant levels. This allows for the specific enhancement of partially reprogrammed MLPSCs while minimizing or reducing the risk of MLPSCs themselves obtaining altered phenotypes or cell identities through premature reprogramming factor expression. In some embodiments, the composition for enhanced partial reprogramming is provided as a pharmaceutical composition comprising genetically modified MLPSCs and pharmaceutically acceptable excipients.

[0174] Exosomes derived from MLPSCs

[0175] In some embodiments, the treatment methods disclosed herein comprise administering purified exosomes or extracellular vesicles isolated from MLPSCs or modified MLPSCs.

[0176] In other embodiments, provided herein are compositions containing exosomes derived from a high-efficiency hMSC population. In some embodiments, such extracellular vesicles or exosomes are generated by preparation from any high-efficiency hMSC population described herein.

[0177] Methods for obtaining clinical-grade extracellular vesicles and exosomes are known in the art. See, for example, Park et al., (2019), Stem Cell Research & Therapy, 10:288; and Yin et al., (2019), Biomarker Research, 7:8.

[0178] For example, exosomes can be isolated from MLPSCs grown to about 70% confluence in serum-containing medium depleted of exosomes over a period of about 24-48 hours. The resulting culture medium is then centrifuged 2-3 times at a low speed (500 x g-800 x g) for 15 minutes to 30 minutes. Afterwards, the resulting supernatant is spun 2-3 times at a higher speed (about 2000 x g to 4000 x g) for about 15 minutes to 30 minutes, and the supernatant from this rotation is collected and spun a third time at about 10,000 x g to 15,000 x g for about 30 minutes. This supernatant is collected and transferred to, for example, an Ultra-Clear centrifuge tube (Beckman Coulter) and centrifuged at 70,000 x g for 1-2 hours at 4 ° C in a SW32Ti rotor (Beckman Coulter) or an equivalent rotor. Carefully decant the supernatant, resuspend the exosome fraction in a physiological saline buffer (e.g., PBS), and repeat the ultracentrifugation cycle. Afterwards, gently resuspend the exosome pellet in about 100 μl to about 500 μl of physiological buffer. If necessary, the resuspended exosomes can be stored at -80°C.

[0179] In an exemplary embodiment of separating the extracellular vesicle part, the MLPSC supernatant obtained as described in the above example is collected and centrifuged at 400 × g-600 × g for 10 minutes, followed by centrifugation at 2000 × g for 20 minutes. In each of these steps, the supernatant is collected and transferred to a new tube for the next step, and the resulting precipitate is discarded. The final supernatant from these low-speed rotations is ultracentrifuged in continuous rotation at the following increasing speeds: at 4 ° C in a 12110 angle rotor (Sigma, 3K30, Germany) or an equivalent rotor with 10,000 × g, 20,000 × g, 40,000 × g and 60,000 × g for about 60 minutes to 90 minutes. The precipitate is then washed about 3 to 4 times with a physiological buffer, and the ultracentrifugation step is repeated. Afterwards, the resulting precipitate can be resuspended in a physiological buffer and used or stored at -80 ° C.

[0180] Administration of MLPSCs, exosomes, or conditioned medium

[0181] MLPSCs (e.g., STRO-1 + Cells, MSCs, their culture-expanded multipotent progeny, their derived exosomes, or MLPSC-conditioned medium) can be administered by any of a number of routes, taking into account the location, distribution, and circulation pathways of the target cell population to be partially reprogrammed and the target tissue in which it resides.

[0182] In some embodiments, MLPSCs are administered to the subject's bloodstream, for example, parenterally. Exemplary parenteral administration routes include, but are not limited to, intra-arterial, intravenous, or intraperitoneal administration routes. In some embodiments, MLPSCs are administered systemically in the case where the target cells to be partially reprogrammed are located in the liver or kidney.

[0183] In some embodiments, in the case where the target cell population is in the heart, administration is performed intra-arterially, into the aorta, into the atrium, or into the ventricle. In the case where the cells are delivered to the atria or ventricles of the heart, the cells can be administered to the left atrium or left ventricle to avoid complications due to rapid delivery of the cells to the lungs. In one embodiment, the group is administered into the carotid artery. In another embodiment, the group is administered into the myocardium. In one example, the group is administered into the inflamed myocardium. In certain instances, the inflamed myocardium is identified using a suitable mapping catheter before the cells are administered to one or more identified sites of inflammation.

[0184] In other embodiments, when the target cells are present in the intervertebral disc, the MLPSCs are administered in the intervertebral disc space or in the nucleus pulposus. In other embodiments, when the target cells are present in the joints, the MLPSCs are administered by intraarticular injection. In other embodiments, when the target cells are located in the brain, the MLPSCs are administered by intracerebral routes, intraventricular routes, intrathecal routes, or by intracarotid routes of administration.

[0185] In other embodiments, administration is intramuscular.In other embodiments, administration of the MLPSCs is intradermal.

[0186] In some embodiments, the administration of MLPSC and or exosomes derived therefrom is completed with a single bolus dose. In other embodiments, administration is performed by continuous infusion or by administration at intervals of, for example, one day, one week, or one to seven times per week. An exemplary dosage regimen is a regimen involving the maximum dose or dosage frequency that avoids significant undesirable side effects. The total weekly dose depends on the type and activity of the factor / cell used. The determination of the appropriate dose is made by a clinician, for example, using parameters or factors known in the art that may affect treatment or predict the success of treatment and / or partial reprogramming of target cells. Typically, the dose starts with an amount slightly below the optimal dose and then increases slightly until the desired or optimal effect is achieved relative to any negative side effects.

[0187] In some embodiments, in the methods disclosed herein, MLPSC or exosomes derived from MLPSC are administered before the initial expression of one or more reprogramming factors to be expressed in the subject. In some embodiments, initial administration is provided between about 21 days to about one hour before the initial expression of one or more reprogramming factors of the subject, for example, 20 days, 18 days, 16 days, 14 days, 12 days, 10 days, 8 days, 7 days, 5 days, 3 days, 2 days, 1 day, 12 hours, 8 hours, 3 hours, or another time point of about 21 days to about one hour before the initial expression of one or more reprogramming factors. In some embodiments, further administration of MLPSC is not provided after the expression of one or more reprogramming factors is initiated. In some embodiments, MLPSC is administered during at least one period of expression of one or more reprogramming factors. In some embodiments, MLPSC is administered within a time period of about 1 day to about 21 days during the expression of one or more reprogramming factors after the initiation of expression of one or more reprogramming factors, for example, 2 days, 3 days, 4 days, 7 days, 10 days, 12 days, 14 days, 18 days, 20 days, or another time period of about 1 day to about 21 days during the expression of one or more reprogramming factors. In other embodiments, MLPSC or exosomes derived therefrom are administered after the time period of expression of one or more reprogramming factors ends and the expression subsides, for example, within a period of about one day to about two weeks after the expression of the reprogramming factors subsides in the target cell population, for example, 2 days, 3 days, 5 days, 7 days, 10 days, 12 days, or another time period of about one day to about two weeks after the expression of the reprogramming factors subsides in the target cell population.

[0188] The appropriate dosage of MLPSCs for use in the partial reprogramming methods disclosed herein will depend on many factors, including but not limited to the route of administration, the target cell population to be partially reprogrammed, the target cell tissue density, the age, sex, weight, and severity of the health condition of the subject being treated.

[0189] In some embodiments, where MLPSCs are to be administered systemically, about 1 x 10 5 MLPSC / kg is about 1x10 8 MLPSC / kg subject body weight. In some embodiments, 1x10 6 MLPSC / kg is about 5x10 6 MLPSC / kg. In some embodiments, 1x10 7 MLPSC / kg is about 5x10 7 MLPSC / kg.

[0190] In other embodiments, the appropriate dose of MLPSCs to be administered is an assessed tissue volume comprising a target cell population to be partially reprogrammed in a tissue volume affected by a health condition (e.g., heart failure, stroke, renal failure, or IVD), e.g., about 2 x 10 6 MLPSC / cm 3 Affected tissue is about 2x 10 7 MLPSC / cm 3 Affected tissue, e.g. 3x 10 6 pcs, 4x 10 6 pcs, 5x 10 6 pcs, 8x 10 6 pcs, 1.2x10 7 pcs, 1.5x10 7 pcs, or from about 2x10 6 MLPSC / cm 3 About 2x 10 7 MLPSC / cm 3 Another cell / cm of affected tissue 3 quantity.

[0191] In other embodiments, dosing is based on the number of MLPSCs to be administered that corresponds to a relative percentage of the number of cells in the target population to be partially reprogrammed, e.g., about 0.5% to about 50% of the number of target cells to be partially reprogrammed, e.g., 1%, 2%, 5%, 7%, 10%, 15%, 20%, 30%, 40%, 45%, or another percentage of the number of target cells to be partially reprogrammed. For example, in a human subject suffering from congestive heart failure and requiring partial reprogramming of cardiomyocytes, the number of MLPSCs administered (e.g., intracardiac administration of MLPSCs) can be about 1% of the total cardiomyocytes in the human heart (about 2 billion to 3 billion cardiomyocytes), i.e., about 2 x 10 7 Up to 3x 10 7 MLPSCs.

[0192] In some embodiments, instead of MLPSC or in addition to MLPSC, isolated exosomes or extracellular vesicles derived from MLPSC (e.g., hMLPSC) are administered to the subject. In some embodiments, extracellular vesicles or exosomes derived from. In some embodiments, the effective amount of extracellular vesicles or exosomes for partial reprogramming is about 50 μg (extracellular vesicle protein or exosome protein content) to about 1,000 μg (extracellular vesicle protein or exosome protein content), such as 60 μg, 70 μg, 80 μg, 100 μg, 250 μg, 300 μg, 400 μg, 500 μg, 600 μg, 800 μg, or about 50 μg (extracellular vesicle protein or exosome protein content) to about 1,000 μg (extracellular vesicle protein or exosome protein content) of extracellular vesicles or exosomes Another amount.

[0193] In some embodiments, MLPSCs or exosomes / extracellular vesicles derived therefrom are administered in a composition comprising a carrier or excipient.

[0194] Suitable carriers for use in the present disclosure include those conventionally used, for example, saline, aqueous dextrose, lactose, Ringer's solution, buffered solutions, hyaluronic acid and glycols are exemplary liquid carriers, particularly (when isotonic) for solutions. In some embodiments, the composition administered includes a pharmaceutically acceptable amount of residual cryopreservative.

[0195] In another example, the carrier is a culture medium composition, such as a cell is grown or suspended in. In one example, such a culture medium composition does not induce any side effects in a subject to which it is administered.

[0196] Exemplary carriers and excipients do not adversely affect cell viability.

[0197] In one example, the carrier or excipient provides buffering activity to maintain cells at a suitable pH to exert biological activity, for example, the carrier or excipient is phosphate buffered saline (PBS). PBS represents an attractive carrier or excipient because it has minimal interaction with cells and factors and allows rapid release of cells and factors, in which case the compositions of the present disclosure can be produced as a liquid for application, for example, by injection, directly to the bloodstream or to tissues or areas surrounding or adjacent to tissues.

[0198] In some embodiments, different subpopulations of MLPSCs are administered in the partial reprogramming methods disclosed herein. In some embodiments, in the methods disclosed herein, a mixture of modified MLPSCs (eg, containing exogenous miRNA) and unmodified MLPSC populations is administered to a subject.

[0199] In some embodiments, different populations of MLPSCs are administered to a subject at different time points. For example, a cell population comprising a target for p16 can be provided before the onset of reprogramming factor expression in the target cell population of the subject. INK4a siRNA was administered to MLPSCs and to unmodified MLPSCs a few days after reprogramming factor expression.

[0200] As will be understood, the enhanced partial reprogramming method described herein can be used for tissue reconstruction or regeneration of human patients in need. For example, the efficacy of enhanced partial reprogramming in spinal cord injury neural cell transplantation can be assessed in a rat model of acute spinal cord injury, as described by McDonald et al. ((1999) Nat. Med., Vol. 5: 1410) and Kim et al. ((2002) Nature, Vol. 418: 50). Successful partial reprogramming will show that new astrocytes, oligodendrocytes and / or neurons migrate along the spinal cord from the damaged end, and improve gait, coordination and weight-bearing capacity.

[0201] Similarly, the efficacy of enhanced partial reprogramming can be evaluated in appropriate animal models of cardiac injury or dysfunction, such as animal models of cardiac hypothermia, in which about 55% of the left ventricular wall tissue becomes scar tissue without treatment (Li et al. (1996), Ann. Thorac. Surg., Vol. 62: 654; Sakai et al. (1999), Ann. Thorac. Surg., Vol. 8: 2074; Sakai et al. (1999), J. Thorac. Cardiovasc. Surg., Vol. 118: 715). Successful treatment will reduce scar area, limit scar expansion, and improve cardiac function, as determined by systolic, diastolic, and developmental pressures (Kehat, et al. (2004)). Cardiac injury can also be simulated, for example, using an embolization coil of the distal portion of the left anterior descending artery (Watanabe et al. (1998), Cell Transplant., Vol. 7: 239), or by ligating the left anterior descending coronary artery (Min, et al. (2002), J. Appl. Physiol., Vol. 92: 288). The efficacy of the treatment can be evaluated by histology and cardiac function. The cardiomyocyte preparations exemplified in the present invention can be used in therapies for regenerating the myocardium and treating cardiac insufficiency.

[0202] Liver function can also be restored by enhanced partial reprogramming using the methods disclosed herein. The results of enhanced partial reprogramming in damaged liver can be evaluated in animal models to assess the ability to repair liver damage.

[0203] The invention is further described in the following non-limiting examples.

[0204] Example

[0205] Example 1: Effect of MLPSC on the partial reprogramming efficiency of fibroblasts using lentivirus

[0206] MLPSC Group

[0207] As an exemplary MLPSC population, STRO-1 + Multipotent progenitor cells (MPCs) were isolated from the bone marrow by immunoselection using STRO3 mAb, then cultured, expanded, and cryopreserved in ProFreezeTM-CDM (Lonza, USA), essentially as described in Gronthos and Zannettino Methods Mol Biol. 449:45-57, 2008). To evaluate the effect of MLPSCs on partial reprogramming efficiency, fibroblasts previously transduced with the reprogramming factors OCT4, KLF4, SOX2, and c-MYC ("OKSM") were co-cultured with allogeneic STRO-1 + MPCs were co-cultured at different MPC to fibroblast ratios. Passage 4 MPCs were thawed and formulated in vehicle for immediate use.

[0208] Fibroblasts

[0209] Primary human skin fibroblast cell lines-GM09503 (passage frozen (P), P3), GM01651 (P14), GM01681 (P12) and GM01680 (P12) were purchased from Coriell Research Institute (Camden, NJ, USA). Fibroblasts were cultured in MEM (Gibco) supplemented with 15% (vol / vol) heat-inactivated FBS (Gibco), 1% (vol / vol) MEM non-essential amino acids (Gibco) and 1% (vol / vol) Pen-Strep (Gibco) in a humidified cell culture incubator (37°C, 5% CO2).

[0210] Effect of MPC on partial reprogramming efficiency of human dermal fibroblasts

[0211] Human dermal fibroblast cultures plated on rh-Laminin-521 (ThermoFisher) were transduced with two lentiviruses: (1) a doxycycline-inducible polycistronic OKSM reprogramming vector; and (2) an EF-2α constitutive rtTA expression vector. After adding polybrene vehicle at a final concentration of about 6 μg / ml (Sigma), the lentiviruses were added to the fibroblast cultures at a multiplicity of infection (MOI) of about 5.

[0212] The next day, the medium containing the lentivirus was replaced with fresh fibroblast medium (10% FBS) containing doxycycline (2 μg / ml), and MPCs were added to a subset of the transduced fibroblast cultures at a ratio of 0.1, 0.5, 1, and 3 MPCs to fibroblasts. After seven days of co-culture in the presence of doxycycline, the expression of the early reprogramming marker TRA-1-60 in the cultures was analyzed by flow cytometry. Co-cultures of MPCs and non-transduced fibroblasts were used as negative controls. TRA-1-60 was expressed in 1:1 ratio in each condition. + The ratio of cells was used as an indicator of relative reprogramming efficiency. It is expected that the presence of MPCs will increase reprogramming efficiency, such as through TRA-1-60 + The proportion of cells.

[0213] Example 2: Effect of MLPSC on mRNA-based partial reprogramming efficiency

[0214] Reprogramming of somatic cells using mRNA-based reprogramming is generally considered to be essentially free of the associated risks of genomic integration / mutation events. Therefore, mRNA-based partial reprogramming may be a safe approach for in vivo / therapeutic applications. Therefore, we evaluated the effects of mRNA-based partial reprogramming of MLPC on aged human dermal fibroblasts in vitro.

[0215] Fibroblasts

[0216] Human adult dermal fibroblasts as described above were cultured in Eagle's Minimum Essential Medium containing Earl's salts supplemented with non-essential amino acids, 10% FBS and 1% penicillin / streptomycin.

[0217] Eagle mRNA transfection and MLPSC co-culture

[0218] Fibroblasts from individuals aged 60-90 years were cultured on iMatrix-511 and transfected with Lipofectamine MessengerMAX using the manufacturer's protocol. TM During the transfection period, use Serum-free medium (Sartorious 05-100-1A). StemRNA TMThe mRNA mixture of encoding reprogramming factors OCT4, SOX2, KL4, c-MYC, NANOG and LIN-28 provided in the third generation reprogramming kit (Reprocell 00-0076). After transfection for the fourth day, the culture medium was replaced with a 1: 1 mixture of StemFit of MSC culture medium (Reprocell AS-MSC) and NutriStem culture medium containing MPC as described in Example 1, so that the final ratio of MPC to fibroblasts was 0.1, 0.5, 1 and 3. Then starting from the initial fibroblast transfection, the co-culture was maintained and passaged for a total of ten days as needed. Afterwards, the expression of early reprogramming marker TRA-1-60 in the culture was analyzed by flow cytometry. The co-culture of MPC and untransduced fibroblasts was used as a negative control. TRA-1-60 under each condition was expressed in 1: 1. + The ratio of cells was used as an indicator of relative reprogramming efficiency. It is expected that the presence of MPCs will increase reprogramming efficiency, such as through TRA-1-60 + The proportion of cells.

[0219] Determination of epigenetic age changes during partial reprogramming

[0220] Epigenetic clocks based on DNA methylation levels are useful molecular biomarkers of age across tissues and cell types, and can predict a range of age-related diseases including life span (Horvath et al. 2013, Genome Biol., 14: R115). Exogenous expression of known canonical reprogramming factors (OSKMs) restores the epigenetic age of primary cells to the prenatal state (Horvath et al., supra). In order to determine the effect of partial reprogramming regulated by MLPSC co-culture, two epigenetic clocks applied to human fibroblasts were used: Horvath's original pan-tissue epigenetic clock (based on 353 cytosine-phosphate-guanine pairs), and the recent skin and blood clock (based on 391 CpGs)-Horvath et al., supra.

[0221] Genomic DNA samples isolated from fibroblast cultures before transfection, and TRA-1-60 at 6, 8, 12, and 15 days after initial transfection +Select, analyze methylation pattern on people Illumina Infinium EPIC 850K chip.Use previously defined mathematical algorithm to combine methylation levels of 353 CpGs into age estimation (in years), which is called epigenetic age or "DNAm age" (Horvath et al., supra).Skin and blood epigenetic clock (based on 391 CpGs) are also used for secondary analysis, because it is known to produce more accurate DNAm age estimation in fibroblasts and several other cell types.The online version of epigenetic clock software is used to reach DNA methylation age estimation.

[0222] Example 3: Effect of MLPSCs on adenovirus-based partial reprogramming of cardiomyocytes in vivo

[0223] Inducible adenovirus for in vivo expression of reprogramming factors

[0224] To perform non-integrating partial reprogramming in vivo, we constructed a regulatable adenovirus for simultaneous expression of the reprogramming factors OCT4, SOX2, KLF4 and c-MYC (“OKSM”) genes and red fluorescent protein (mCherry) based on the construct described in Lehmann et al., (2019), Gene Therapy, 26:432-440.

[0225] Adenoviral vectors were constructed using a commercial kit (Microbix Inc., Ontario, Canada) that provides the shuttle plasmid pC4HSU, helper adenovirus H14, and HEK293Cre4 cell line. As a starting point, we used a regulatable Tet-On bidirectional construct (pTRE3G-BI-mCherry; Takara catalog number 631333) that placed the mCherry gene flanked by a bidirectional promoter P min CMV-TRE-P minOne of two multiple cloning sites (MCS) of CMV. On the separate site of the construct, a constitutive expression cassette (CMV-rtTA) expressing DOX activation ("reverse tetracycline transactivator") rtTA was cloned. In the second MCS, we cloned OCT4-f2A-KLF4-IRES-SOX2-p2A-Cmyc in a bicistronic series. The encoded reprogramming factors are grouped in pairs, located downstream and upstream of the internal ribosome entry site (IRES), and each pair of reprogramming factor ORFs is separated by a 2A type CHYSEL (cis-acting hydrolase element) self-processing short sequence, which causes the ribosome to skip the Gly-Pro bond at the C-terminal end of the 2A sequence, thereby releasing the peptide upstream of the 2A element but continuing the translation of the downstream mRNA sequence. This allows the co-expression of two cistrons of the 2A type sequence flanking the stoichiometric. For reference, this construct is called prtTA-TRE-OKSM-mCherry.

[0226] Next, the entire bidirectional TRE-OKSM-mCherry expression cassette is excised and cloned into the pC4HSU shuttle vector to produce pC4HSU-rtTA-TRE-OKSM-mCherry. The pC4HSU shuttle vector consists of the reverse terminal repeats (ITRs) of the Ad 5 virus, packaging signals, and part of the E4 adenovirus region plus a filler non-coding DNA of human origin, which keeps the appropriate size (28-31Kbp) of the viral DNA. There is a bacterial sequence flanked by a Pme I site between the two ITRs. The pC4HSU-rtTA-TRE-OKSM-mCherry plasmid is digested with Pme I to remove the bacterial sequence, thereby producing the desired HD-RAd-rtTA-TRE-OKSM-mCherry genome ("Ad reprogramming").

[0227] The linearized DNA backbone of the Ad reprogramming construct is transfected into Cre 293 cells. On the second day, the purified helper H14 virus is added to the cell culture with an MOI of 5. In H14, the packaging signal is flanked by lox P sites, which are recognized by the Cre recombinase expressed by 293Cre4 cells. Therefore, the helper virus trans provides all the viral products required for generating the desired Ad reprogramming virus. The infected 293Cre4 cells are retained for 2-3 days until cytopathic effect (CPE) occurs. Cells and culture medium are collected and three freeze-thaw cycles are performed to lyse them. Clarified lysate is obtained, mixed with H14 helper virus, and added to the fresh culture of Cre4 293 cells with an MOI of 1. When CPE occurs, the second generation (P2) cell lysate is prepared. This iterative co-infection process is repeated five more times to generate a sufficient number of adenovirus particles for purification steps. The newly generated adenovirus particles are rescued from P6. The Ad reprogramming virus particles generated in this way are purified by ultracentrifugation on a CsCl gradient. The final viral stock was titrated by lysing viral particles, extracting their DNA and measuring their concentration in a Nanodrop spectrophotometer. The titer after purification was approximately 1 × 10 12 Virus particles / ml. A control adenovirus with Dox-inducible EGFP (Ad-EGFP) was used as a negative control as described below.

[0228] Virus stocks were resuspended in phosphate-buffered saline (PBS) containing 5% glycerol and diluted in normal saline before injection.

[0229] Ad reprogramming and intramyocardial injection of MLPSCs

[0230] Groups 1-5 of thymic nude mice (n=6 per group) received left anterior descending coronary artery (LAD) ligation to induce myocardial infarction. Two days later ("Day 0"), groups (1-5) of animals as shown in Table 1 below received sham injections (Groups 1 and 2), injections of 1 μl (1 x 10 9 Virus particles), Ad reprogramming (Groups 3 and 4) or Ad-EGFP (Groups 5 and 6). The next day ("Day 1"), drinking water was provided to the animal groups listed below, and the drinking water contained 0.2 mg / ml doxycycline hydrochloride supplemented with 7.5% sucrose (Groups 3-6); or only 7.5% sucrose (Groups 1 and 2) for a period of 7 days. The second day ("Day 2") of inducing the expression of reprogramming factors, 1x 106 MLPSC or sham injection (1, 3) was administered to each designated group by direct intramyocardial injection (2, 4, 6). The overall systolic and diastolic parameters of cardiac function were determined on days 10-15. On day 16, animals were sacrificed and new blood vessel formation and immunostaining and cytometry were performed for cardiomyocytes and co-localized mitotic markers.

[0231]

[0232] Example 4: Serum Analysis

[0233] Mesenchymal precursor lineage or stem cell populations were cultured and expanded in 5% FCS / 5% NBCS (Serum A) or 10% fetal bovine serum (Serum B). These MLPSCs were used in Example 7.

[0234] Cytokine levels were assessed in 5% FCS / 5% NBCS (Serum A) and 10% fetal bovine serum (Serum B). To provide external controls, cytokine levels were also assessed in FBS from different suppliers (Serum C). In each case, cytokine levels were assessed in pure serum.

[0235] Surprisingly, serum preparations containing newborn calf serum had higher levels of proinflammatory cytokines (Figure 1). Notably, there was an increase in proinflammatory cytokines known to bind to receptors expressed on the surface of MLPSCs, such as interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and interleukins. For example, the following was observed in serum preparations containing newborn calf serum relative to fetal bovine serum:

[0236] IFNγ increased by at least 2-fold;

[0237] TNFα increased by at least 13-fold;

[0238] IL-6 increased at least 8-fold;

[0239] IL-8 increased by at least 2-fold;

[0240] IL-17A increased at least 2-fold.

[0241] Example 5: MLPSC compositions derived using culture medium containing fetal serum

[0242] The alpha modification of Eagle's Minimum Essential Medium (MEM) with Earle's Balanced Salts (commonly referred to as Eagle alpha MEM) contains nonessential amino acids, sodium pyruvate, and additional vitamins. These modifications were originally described for growing hybrid mouse and hamster cells (Stanners et al. 1971).

[0243] Eagle αMEM medium suitable for culturing primary stem cells is available from a variety of sources, including Life Technologies and Sigma.

[0244] Detailed methods for establishing primary stem cell cultures, including the required growth factors used in the exemplified procedures, are described in Gronthos and Simmons 1995.

[0245] The cells were supplemented with 10% fetal bovine serum, L-ascorbic acid-2-phosphate (100 μM), dexamethasone (10 -7 MLPSCs were cultured in Eagle αMEM medium supplemented with 1% glutathione (M) and / or inorganic phosphate (3 mM).

[0246] Example 6: Pre-licensed culture medium (neonatal serum medium)

[0247] For MLPSC medium containing neonatal serum, the serum component of Eagle αMEM medium described in Example 2 was modified by supplementing with 5% (volume / volume) neonatal serum (the difference between fetal serum medium and neonatal serum medium is shown in Table 2). The neonatal serum used was newborn calf serum (NBCS; serum A). NBCS is 100% bovine serum obtained from animals that meet standard fetal bovine serum specifications but are less than 20 days old.

[0248] NBCS was obtained from a commercial supplier where it is marketed as an FCS replacement, is highly similar to FCS, can be used interchangeably, and is expected to have the same effect on cell lines.

[0249] Table 2: Summary of differences between fetal serum media and licensed media

[0250]

[0251] Example 7: Comparison of MPCs cultured in medium supplemented with neonatal serum and cultured in medium containing fetal serum alone Evaluation of cultured MPCs in vitro: therapeutic efficacy in the context of persistent inflammation

[0252] NYHA class II / III high-risk heart failure with reduced ejection fraction (HFrEF) is a clinical model of persistent inflammation. HFrEF patients are characterized by cardiac and systemic inflammation, as determined by the presence of elevated inflammatory biomarkers. In the following clinical study, MPCs cultured under different serum conditions were administered to HFrEF patients. In HFrEF patients, cardiac macrophages produce high levels of proinflammatory cytokines (IL-6, IL-1, TNF-α), which lead to endothelial dysfunction and cardiomyocyte apoptosis. Plasma C-reactive protein (CRP) levels, as determined by high-sensitivity CRP (hsCRP) assays, reflect the acute phase reactants produced by the liver in response to high levels of proinflammatory cytokines (IL-6, IL-1, and TNF-α) produced by cardiac macrophages. Therefore, plasma hsCRP levels (<2 mg / L vs. >2 mg / L) are representative systemic measurements reflecting low or high intracardiac inflammation. In subsequent studies, HFrEF patients were classified as having persistent inflammation if their plasma hsCRP levels were >2 mg / L.

[0253] Study Details

[0254] Eligible NYHA class II / III patients were enrolled in a double-blind, randomized, sham-controlled, parallel-group efficacy and safety study of allogeneic mesenchymal precursor cells (Rexlemestrocel-L) in the DREAM HF-1 trial in chronic heart failure caused by LV systolic dysfunction (ischemic or non-ischemic). HFrEF patients were administered: (1) MPCs cultured in 10% fetal serum (n=37), (2) MPCs cultured in the presence of newborn calf serum (5% FCS / 5% NBCS; n=153), or (3) sham controls (i.e., no MPCs; n=241). Cells cultured in medium supplemented with newborn serum were effectively cultured in medium containing increased levels of proinflammatory cytokines, as demonstrated by serum analysis described in Example 4. Cells were administered by a single transendocardial injection. LV systolic function in HFrEF was measured by echocardiographic (ECHO) parameters including left ventricular ejection fraction (LVEF; %), left ventricular end-systolic volume (LVESV; mL), and left ventricular end-diastolic volume (LVEDV; mL) at baseline and after 12 months of treatment. Plasma CRP levels were measured to determine baseline levels of inflammation.

[0255] result

[0256] MPCs cultured in the presence of neonatal calf serum (5% FCS / 5% NBCS) were found to improve left ventricular (LV) systolic function in HFrEF patients at 12 months. Specifically, MPCs cultured in neonatal serum significantly increased LVEF and decreased LVESV compared with sham controls (p = 0.0398 and 0.0426, respectively) ( Figure 2 ).

[0257] HFrEF patients were then characterized based on plasma hsCRP levels <2 mg / L (normal baseline systemic inflammation) or >2 mg / L (elevated baseline systemic inflammation). Importantly, when HFrEF patients were differentiated based on baseline systemic inflammation status (CRP>2), the effect of MPCs cultured in the presence of newborn calf serum (5% FCS / 5% NBCS) in inducing recovery of LV systolic function was more pronounced. In contrast, MPCs cultured in 10% FBS did not induce a significant effect ( Figure 3Compared with sham controls, MPCs cultured in neonatal serum significantly increased LVEF% by a mean (LS mean) of 2.46 and decreased LVESV by a mean of 8.99 mL (p = 0.0033 and 0.0264, respectively). In contrast, MPCs cultured in 10% fetal serum or 5% / FCS / 5% NBCS showed improvements in LV systolic function in HFrEF patients without elevated baseline inflammation (HFrEF patients with CRP < 2). Figure 4 ).

[0258] MPCs cultured in neonatal serum were also found to reduce other cardiac outcomes in HFrEF patients with CRP>2, including a 43% reduction in the risk of cardiovascular death ( Figure 5 ) and 54% 3-point MACE (CV death / MI / stroke) rate ( Figure 6 These data suggest that MPCs cultured in neonatal serum offer improved therapeutic efficacy in the setting of persistent inflammation.

[0259] Further analysis of clinical response surprisingly revealed the importance of culturing and expanding MLPSC in a medium supplemented with neonatal serum and / or proinflammatory cytokines during the final passage before administration. MLPSC cultured in a medium supplemented with neonatal serum and / or proinflammatory cytokines during the final passage reduced the patient's 3-point MACE (MI, stroke or CV death), regardless of whether MLPSC was cultured and expanded in FBS during early passage. Surprisingly, this reduction in 3-point MACE was observed in all patients regardless of the inflammatory state (Figure 10). Patient subgroup analysis revealed that a 3-point MACE reduction was observed in patients with persistent inflammation (CRP>2mg / ml; Figure 7) and in patients with the highest risk of terminal cardiac events (TCE; defined as CV death, heart transplantation or left ventricular assist device implantation) in the study. Patients with the highest risk of TCE are defined as CRP>2mg / ml and NT-proBNP>1000ng / ml; Figure 8)).

[0260] In contrast, an increase in adverse events (3-point MACE) was observed in all patients administered MLPSCs that were cultured and expanded in a medium not supplemented with neonatal serum and / or proinflammatory cytokines during the final passage prior to administration. An increase in 3-point MACE was observed regardless of whether MLPSCs were cultured in a medium supplemented with neonatal serum and / or proinflammatory cytokines during early passages.

[0261] Summarize

[0262] MPCs expanded in medium supplemented with newborn calf serum (NBCS) and / or pro-inflammatory cytokines:

[0263] Improved left ventricular systolic dysfunction in HFrEF patients with inflammation, as measured by LS mean change in LVEF and LVESV at 12 months;

[0264] Reduced cardiovascular death by 43% in high-risk NYHA class II / III patients with HFrEF and inflammation; and

[0265] Reduced long-term 3-point MACE by 54% in high-risk NYHA class II / III patients with HFrEF and inflammation.

[0266] Taken together with the results of Example 4, these human experimental data suggest that supplementing the cell culture medium with neonatal serum and / or proinflammatory cytokines provides cell populations with distinct functional characteristics, at least in terms of the ability to direct therapeutic efficacy in an inflammatory setting.

[0267] Without wishing to be bound by any particular theory, these data suggest that supplementation of media with added pro-inflammatory cytokines and / or NCBS enables MPCs to respond more effectively to the inflammatory milieu and promote partial reprogramming of cells in vivo, consistent with the ability of MPCs to inhibit NK cell activation and NK-mediated partial reprogramming.

[0268] The inventors' findings support the broad application of pre-competent MPCs (e.g., MPCs cultured in medium supplemented with pro-inflammatory cytokines and / or NCBS) for enhanced partial reprogramming and for treating any disease or condition characterized by elevated inflammation, particularly diseases characterized by persistent inflammation (such as heart failure).

[0269] Example 8: MLPSCs cultured in medium supplemented with newborn fetal serum are effective in treating GvHD

[0270] 10 GvHD patients were given 2x 10 6 MPCs expanded with NBCS culture containing proinflammatory cytokines (Examples 4 and 6) were administered at a dose of 100 mg / kg (intravenously). Patient responses are summarized in Table 3. 80% of GvHD patients administered MPCs cultured in NBCS responded to treatment. One patient achieved a complete response, seven patients achieved a partial response and two patients died.

[0271] Table 3. GvHD response to MPCs expanded using NBCS culture.

[0272]

[0273]

[0274] Combined with at least the results of Example 5, these data further demonstrate the anti-inflammatory properties of MLPSC in T cell-mediated disorders. Therefore, there is strong evidence to support the use of MLPSC for partial reprogramming, particularly culturing expanded MLPSC in medium supplemented with proinflammatory cytokines and / or neonatal serum.

Claims

1. A method for enhancing partial reprogramming of target cells in a subject in need thereof, the method comprising administering to the subject a plurality of mesenchymal lineage progenitor or stem cells (MLPSC), exosomes derived therefrom, or conditioned medium derived from a culture of a plurality of MLPSC, wherein the subject expresses or will express one or more reprogramming factors in a target cell population after the administration, thereby causing a plurality of the target cells in the subject to be partially reprogrammed. 2 . The method of claim 1 , wherein the target cell population comprises one or more exogenous nucleic acids encoding the one or more reprogramming factors.

3. The method of claim 1, wherein the target cell population comprises one or more exogenous nucleic acids encoding one or more targeted transactivators for inducing expression of endogenous genes encoding the one or more reprogramming factors.

4. The method according to any one of claims 1 to 3, wherein the MLPSC is derived from STRO-1 + Mesenchymal progenitor cell (MPC) or mesenchymal stem cell (MSC) population culture expansion.

5. The method according to any one of claims 1 to 4, wherein the MLPSC is a pre-licensed MLPSC.

6. The method according to claim 5, wherein the MLPSCs are cultured and expanded in a culture medium containing the following components: -IFN-γ and / or TNF-α; and - one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1-α, MIP-1-β and IP-10.

7. The method of claim 6, wherein the culture medium contains serum comprising IFN-γ and / or TNF-α; and the one or more pro-inflammatory cytokines.

8. The method of claim 7, wherein the culture medium comprises newborn mammalian serum.

9. The method of claim 8, wherein the newborn mammalian serum is newborn calf serum.

10. The method according to any one of claims 1 to 9, wherein the MLPSC is a MLPSC genetically modified to overexpress indoleamine 2,3-dioxygenase (IDO) and / or cyclooxygenase-2 (COX-2).

11. The method according to any one of claims 1 to 10, comprising administering the plurality of MLPSCs.

12. The method of any one of claims 1 to 10, comprising administering the exosomes derived from a plurality of MLPSCs.

13. The method of any one of claims 1 to 10, comprising administering the conditioned medium derived from a plurality of MLPSC cultures.

14. The method of any one of claims 1 to 13, wherein the subject suffers from or is at risk of suffering from a health condition selected from the group consisting of cardiovascular disease, vascular endothelial disorders, low back pain, inflammatory diseases, osteoarthritis, metabolic diseases, kidney disease, liver disease, and neurological disorders.

15. The method according to any one of claims 1 to 14, wherein the target cell is selected from the group consisting of: cardiomyocytes, nucleus pulposus cells, kidney cells, hepatocytes, endothelial progenitor cells, chondroprogenitor cells, chondrocytes, neurons, neural progenitor cells and glial cells.

16. The method according to any one of claims 1 to 15, wherein the one or more reprogramming factors are selected from the group consisting of: OCT4, SOX2, KLF4, c-MYC, LIN28 and NANOG.

17. The method according to any one of claims 1 to 16, wherein the one or more reprogramming factors comprise OCT4, SOX2 and KLF4.

18. The method of claim 17, wherein the one or more reprogramming factors comprise OCT4, SOX2, KLF4 and c-MYC.

19. The method of claim 17, wherein the one or more reprogramming factors comprise OCT4, SOX2, and KLF4 but do not comprise c-MYC.

20. The method of claim 16, wherein the one or more reprogramming factors: (i) do not comprise LIN 28; or (ii) do not comprise NANOG.

21. The method of claim 16, wherein the one or more reprogramming factors comprise OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG.

22. The method of claim 21, wherein the one or more reprogramming factors consist of: (i) OCT4, SOX2, KLF4, c-MYC, LIN28, and NANOG; (ii) OCT4, SOX2, KLF4 and c-MYC; or (iii) OCT4, SOX2 and KLF4.

23. The method of any one of claims 2 to 22, wherein the one or more exogenous nucleic acids comprise one or more synthetic mRNAs encoding the one or more reprogramming factors or encoding the one or more targeted transactivators.

24. The method of claim 23, wherein the one or more synthetic mRNAs comprise one or more nucleoside-modified mRNAs.

25. The method of any one of claims 2 to 22, wherein the one or more exogenous nucleic acids comprise one or more non-integrating expression cassettes for expressing the one or more encoded reprogramming factors or targeted transactivators in mammalian cells.

26. The method of claim 25, wherein the one or more non-integrating expression cassettes are provided as one or more of: a plasmid expression vector, a minicircle expression vector, a linear amplicon, a circular DNA amplicon, a recombinant DNA viral genome or amplicon or a recombinant RNA viral genome, amplicon, or any combination thereof.

27. The method of claim 25 or claim 26, wherein expression of at least one of the one or more encoded reprogramming factors or targeted transactivators is under the control of a target cell selective promoter.

28. The method of claim 27, wherein the target cell selective promoter is selected from the group consisting of a cardiomyocyte selective promoter, a nucleus pulposus cell selective promoter, a kidney cell selective promoter, a hepatocyte selective promoter, an endothelial progenitor cell selective promoter, a chondroprogenitor cell selective promoter, a glial cell selective promoter, a neural progenitor cell selective promoter, and a neuron selective promoter.

29. The method of any one of claims 2 to 28, wherein at least one of the exogenous nucleic acids is a polycistronic.

30. The method according to any one of claims 1 to 29, wherein expression of the one or more reprogramming factors is inducible and reversible.

31. The method of any one of claims 1 to 30, wherein the MLPSC is a modified MLPSC comprising at least one of the following: an exogenous anti-inflammatory agent: miRNA, antagomiR, siRNA, RNAi, antisense oligonucleotide or antisense RNA.

32. The method according to any one of claims 1 to 31, wherein the MLPSC is a modified MLPSC comprising a gene targeting p16 INK4a 、p21 WAF1 / CIP1 , mTOR signaling pathway, or c-Jun N-terminal kinase (JNK) signaling pathway.

33. The method of any one of claims 1 to 32, wherein the one or more reprogramming factors are expressed continuously for at least about two days but no longer than about 15 days.

34. The method of claim 33, wherein the one or more reprogramming factors are expressed for a cumulative period of at least two days but no longer than about 15 days.

35. The method of any one of claims 1 to 34, wherein the plurality of MLPSCs are administered prior to initiating expression of the one or more reprogramming factors.

36. The method of any one of claims 1 to 34, wherein the plurality of MLPSCs are administered after the one or more reprogramming factors have begun to be expressed.

37. The method of any one of claims 2 to 36, further comprising delivering the one or more exogenous nucleic acids to the target cell population.

38. The method of any one of claims 1 to 37, further comprising administering prostaglandin E2 to the subject.

39. The method of any one of claims 1 to 38, further comprising administering a blocking antibody directed against the natural killer cell receptor NKG2D.

40. A method for enhancing partial reprogramming of target cells in a postnatal subject in need thereof, the method comprising administering to the subject a plurality of mesenchymal lineage progenitor or stem cells (MLPSCs), exosomes derived therefrom, or conditioned medium derived from a culture of the plurality of MLPSCs, wherein the subject expresses exogenous reprogramming factors comprising OCT4, SOX2, KLF4, and c-MYC in a target cell population simultaneously with or after administration, wherein one or more exogenous nucleic acids for expressing the exogenous reprogramming factors have been delivered to the target cell population (i) as one or more synthetic mRNAs; (ii) by one or more non-integrating recombinant viruses; or (iii) as a combination of (i) and (ii); whereby a plurality of the target cells in the postnatal subject are partially reprogrammed.

41. The method of claim 40, wherein the one or more exogenous nucleic acids are delivered to the target cell population as one or more synthetic mRNAs.

42. The method of claim 40, wherein the one or more exogenous nucleic acids are delivered to the target cell population via one or more non-integrating recombinant viruses.

43. The method of claim 42, wherein the non-integrating recombinant virus is selected from the group consisting of adenovirus, adeno-associated virus, non-integrating lentivirus, human cytomegalovirus (CMV), herpes simplex virus (HSV), and Sendai virus.

44. The method of any one of claims 40 to 43, wherein the MLPSCs are administered simultaneously with expression of the exogenous reprogramming factors.

45. The method of any one of claims 40 to 43, wherein the MLPSCs are administered prior to expression of the exogenous reprogramming factors.

46. ​​The method of any one of claims 40 to 45, further comprising delivering the one or more exogenous nucleic acids to the target cell population to express the exogenous reprogramming factors.

47. The method of any one of claims 40 to 46, wherein the MLPSC is a pre-licensed MLPSC.

48. The method according to claim 47, wherein the MLPSCs are MLPSCs expanded by culturing in a culture medium containing the following components: -IFN-γ and / or TNF-α; and, - one or more pro-inflammatory cytokines selected from the group consisting of IL-6, IL-8, IL-17A, MCP-1-α, MIP-1-β and IP-10.

49. The method of claim 48, wherein the culture medium contains serum comprising IFN-γ and / or TNF-α; and the one or more pro-inflammatory cytokines.

50. The method of claim 49, wherein the serum is newborn mammalian serum.

51. The method of claim 50, wherein the newborn mammalian serum is newborn calf serum.

52. The method of any one of claims 40 to 51, wherein the MLPSC is administered by a topical administration route.

53. A population of MLPSCs when used in a method according to any one of claims 1 to 52.

54. A composition for enhancing partial reprogramming of a target cell, comprising a plurality of modified mesenchymal lineage progenitor or stem cells (MLPSCs), the MLPSCs comprising one or more exogenous nucleic acids encoding one or more of: (a) a reprogramming factor; or (b) a targeted transactivator that induces expression of one or more endogenous reprogramming factors, wherein: (i) when one or more target cells are present, the modified MLPSC delivers the one or more exogenous nucleic acids to the one or more target cells; (ii) the one or more encoded reprogramming factors are expressed in the one or more target cells at a level sufficient to partially reprogram the one or more target cells; and (iii) the one or more encoded reprogramming factors or targeted transactivators are substantially incapable of expression in the modified MLPSCs prior to the delivering in (i).

55. A composition for enhancing partial reprogramming of target cells, comprising: (i) culture-expanded human mesenchymal stem cell (hMSC) populations; (ii) a conditional replication helper virus; and (iii) one or more helper-dependent viruses, which: (a) contain one or more expression cassettes of one or more reprogramming factors or targeted transactivators that induce expression of one or more endogenous reprogramming factors; and (b) are substantially free of viral coding sequences.

56. The composition of claim 55, wherein the hMSCs are transduced using (ii), (iii), or both (ii) and (iii).

57. The composition of claim 55 or claim 56, wherein the one or more expression cassettes are inducible and reversible expression cassettes.

58. The composition of claim 57, wherein the conditionally replicating helper virus or the one or more helper-dependent viruses encode a regulatable transactivator that controls expression of the one or more inducible and reversible expression cassettes.

59. The composition of any one of claims 55 to 58, wherein the conditional replicating helper virus and the one or more helper-dependent viruses are adenovirus, herpes simplex virus (HSV), non-integrating lentivirus, or adeno-associated virus (AAV).

60. The composition of claim 59, wherein the conditional replication helper virus and the one or more helper-dependent viruses are adenoviruses.

61. The composition of any one of claims 55 to 60, wherein the culture-expanded hMSCs are pre-permitted hMSCs.

62. A pharmaceutical composition comprising a compound according to any one of claims 54 to 61 and a pharmaceutically acceptable excipient.

63. The pharmaceutical composition of claim 62, further comprising one or more of prostaglandin E2 or a blocking antibody against the natural killer cell receptor NKG2D.

64. A method for enhancing partial reprogramming of a target cell, the method comprising administering to a subject in need thereof a composition according to any one of claims 56 to 63.

65. The method of claim 64, wherein the subject suffers from or is at risk for a health condition selected from the group consisting of cardiovascular disease, endothelial disorders, low back pain, inflammatory diseases, osteoarthritis, metabolic diseases, kidney disease, liver disease, and neurological disorders.

66. The method of claim 64 or claim 65, wherein the transduced hMSCs are administered via a topical administration route.

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