Genetically engineered mesenchymal stem cells and uses thereof

Abstract

Therefore, this disclosure provides a genetically engineered mesenchymal stem cell (MSC) population, including an expression vector containing the Akt or HGF gene and the PD-L1 gene. It also provides a method for synergistically improving the survival status and immunomodulatory capacity of mesenchymal stem cells or enhancing their proliferation, including transfecting mesenchymal stem cells with the Akt or HGF gene and the PD-L1 gene; and a method for preventing, improving, and / or treating ischemic conditions, enhancing neurogenesis, or reducing neuronal death, including administering an effective amount of the genetically engineered mesenchymal stem cell population of this disclosure to individuals in need.

Description

Technical Field

[0001] This invention relates to the field of engineered stem cells and their applications. In particular, the engineered stem cells contain at least one survival gene and one immune checkpoint gene, and can be used to treat or improve ischemic conditions, enhance nerve regeneration, or reduce neuronal death. Background Technology

[0002] Stroke and acute myocardial ischemia (AMI) are among the leading causes of death and disability worldwide. Although recombinant tissue plasminogen activator (rt-PA) is a recognized and widely used treatment for acute ischemic stroke and acute myocardial ischemia, its narrow time window limits the benefits of thrombolytic therapy. A novel treatment method with significantly improved efficacy or benefit is needed.

[0003] Mesenchymal stem cells (MSCs) possess broad immunosuppressive potential, capable of modulating the activity of both innate and adaptive immune system cells, and are therefore considered to have the potential to treat a wide range of diseases. However, understanding the behavior of MSCs, and their ability to be utilized to aid in treatment and related applications, remains elusive. Many therapeutic approaches involving MSCs have been tested, but with limited efficacy.

[0004] Therefore, improving the survival rate of mesenchymal stem cells is urgently needed for their therapeutic applications. Summary of the Invention

[0005] This disclosure reveals genetically engineered mesenchymal stem cells (MSCs) based on elevated levels of Akt or hepatocyte growth factor (HGF) and PD-L1, and methods for using these MSCs to treat and improve ischemic conditions, enhance neurogenesis, or reduce neuronal death. The mesenchymal stem cells and methods disclosed herein lead to significant and unexpected improvements in injury recovery and repair in individuals requiring treatment.

[0006] In one aspect, this disclosure provides a method for preventing, improving, and / or treating ischemic conditions, enhancing neurogenesis, or reducing neuronal death in an individual, comprising administering to the individual an effective amount of a mesenchymal stem cell population containing survival genes (such as Akt or HGF) and immune checkpoint genes (such as PD-L1). The use of the mesenchymal stem cell population containing survival genes (such as Akt or HGF) and immune checkpoint genes (e.g., PD-L1) in the manufacture of a pharmaceutical agent for treating or improving ischemic conditions in an individual is also provided. In one embodiment, the mesenchymal stem cells are genetically engineered.

[0007] Examples of ischemic conditions include, but are not limited to, stroke and myocardial infarction (MI). In one embodiment, the MI is acute myocardial infarction (AMI). In one embodiment, the application of this disclosure reduced MI-induced fibrosis. In another embodiment, the application of this disclosure also reduced inflammation in ischemic tissue. In another embodiment, the application of this disclosure reduced left ventricular dysfunction after MI and reduced infarct size after MI. In another embodiment, the application of this disclosure increased the expression of regulatory molecules on T cells in the spleen after stroke, reduced neuronal death and / or reduced inflammatory response caused by stroke brain injury, but enhanced CD8+ expression in the ischemic brain. + CD122 + Accumulation of Tregs.

[0008] In one implementation, the application reduced the inflammatory response but enhanced CD8+ in ischemic tissue. + CD122 + Accumulation of Tregs. Preferably, the ischemic tissue is ischemic brain tissue.

[0009] In one implementation, this administration increased the expression of regulatory molecules in individual T cells.

[0010] In one embodiment, the effective amount of genetically engineered MSCs revealed in this study increased the expression of Akt or HGF and PD-L1.

[0011] The effective quantity of the genetically engineered MSCs population disclosed herein, for some embodiments, ranges from approximately 1 × 10⁻⁶. 5 From one cell to approximately 1 × 10 8 The effective quantity of this genetically engineered MSC population ranges from approximately 3 × 10⁻⁶ cells in certain embodiments. 5 One cell to approximately 3 × 10 8 1 cell, approximately 5 × 10 5 Cells to approximately 5 × 10⁸ cells, approximately 7 × 10⁸ cells 5 Cells to approximately 7 × 10 8 Cells, approximately 1 × 10 6 Cells to approximately 1×10 8 Cells, approximately 3 × 10 6 Cells to approximately 3 × 10 8 Cells, approximately 5 × 10 6 Cells to approximately 5 × 10 8 Cells, approximately 7 × 10 6 Cells to approximately 7 × 10 8 Cells, approximately 1 × 10 7 Cells to approximately 1×10 8Cells, approximately 3 × 10 7 Cells to approximately 3 × 10 8 Cells, approximately 5 × 10 7 Cells to approximately 5 × 10 8 Cells, approximately 7 × 10 7 Cells to approximately 7 × 10 8 Cells, approximately 1 × 10 5 Cells to approximately 1×10 6 Cells, approximately 3 × 10 5 Cells to approximately 3 × 10 6 Cells, approximately 5 × 10 5 Cells to approximately 5 × 10 6 Cells or approximately 7 × 10 5 Cells to approximately 7 × 10 6 cell.

[0012] In some embodiments, administration includes, but is not limited to, intravenous injection, intracarotid injection, intra-arterial injection, or combinations thereof. One embodiment of administration is intracarotid injection combined with intravenous injection. Another embodiment of administration is intra-arterial injection combined with intravenous injection. Preferably, intravenous injection is performed before intra-arterial injection. In one particular embodiment, in individuals with stroke or AMI, administration is either intracarotid injection combined with intravenous injection or intra-arterial injection combined with intravenous injection. In another particular embodiment, the effective dose range is approximately 1 × 10⁻⁶ for intracarotid injection. 4 Cells to approximately 1×10 6 Cells, preferably about 5 × 10 4 Cells to approximately 5 × 10 5 Cells, the effective dose range for intravenous injection is approximately 3 × 10⁻⁶. 4 Cells to approximately 1×10 7 Cells, preferably about 1 × 10 5 Cells to approximately 5 × 10 6 cell.

[0013] On the other hand, this disclosure provides a method for synergistically increasing the survival status and immunomodulatory capacity of MSCs or enhancing MSC proliferation, including transfecting mesenchymal stem cells with Akt or HGF genes and PD-L1 genes.

[0014] In one aspect, this disclosure provides a population of genetically engineered mesenchymal stem cells (MSCs), wherein the MSCs contain survival genes (e.g., Akt or HGF) and immune checkpoint genes (e.g., PD-L1). In one embodiment, the mesenchymal stem cells are genetically engineered.

[0015] In one embodiment, the mesenchymal stem cells in this paper are selected from a group consisting of umbilical cord mesenchymal stem cells (UMSCs), adipose-derived mesenchymal stem cells (ADSCs), and bone marrow mesenchymal stem cells (BMSCs).

[0016] In one embodiment, the programmed death-ligand 1 (PD-1), Akt, or HGF described herein is transduced using a transposon or vector. In some embodiments, the survival gene (e.g., Akt or HGF) and the immune checkpoint gene (e.g., PD-L1) are contained in an expression vector. In one embodiment, the expression vector is a viral vector. In another embodiment, the viral vector is a lentiviral vector.

[0017] On the other hand, this disclosure provides a pharmaceutical composition comprising the genetically engineered MSCs population disclosed herein.

[0018] Simple Explanation of the Diagram

[0019] Figure 1A This image displays the results of morphological observations of primary cultures of umbilical cord mesenchymal stem cells (UMSCs).

[0020] Figure 1B This image shows the results of flow cytometry analysis of the biological characteristics of primary cultures of umbilical cord mesenchymal stem cells.

[0021] Figure 1C This demonstrates the use of flow cytometry to detect RFP fluorescence of UMSC-PD-L1-Akt and the expression level of PD-L1.

[0022] Figure 1D This study demonstrates the detection of Akt expression levels in UMSC-PD-L1-Akt using the Western ink dot method.

[0023] Figure 1E Microscopic observation results of adipogenesis, chondrogenesis, osteogenic formation, and vascularization in UMSC-Akt-PD-L1 and ordinary UMSC.

[0024] Figure 2A The image shows the proliferation results of UMSC-PD-L1-Akt and ordinary UMSCs detected by CFSE staining.

[0025] Figure 2BThe results show the cell viability of UMSC-PD-L1-Akt, UMSC-Luc and UMSCs treated with H2O2.

[0026] Figure 2C The graph shows the cell proportions of TUNEL+ after H2O2 treatment of UMSC-PD-L1-Akt, UMSC-Luc, and UMSCs.

[0027] Figure 2D The image shows the results of DCF fluorescence analysis of intracellular reactive oxygen species levels after H2O2 treatment of UMSC-PD-L1-Akt, UMSC-Luc, and UMSCs.

[0028] Figure 2E The results of H2O2 treatment of UMSC-PD-L1-Akt, UMSC-Luc and UMSCs are shown in the graph of Akt phosphorylation by Western ink dot analysis.

[0029] Figure 3A The image shows the results of intravenous injection of IgG, UMSCs, UMSC-Akt, UMSC-HGF, UMSC-PD-L1, UMSC-PD-L1-Akt, or UMSC-PD-L1-HSF in the post-stroke infarct area.

[0030] Figure 3B The image shows the results of intracarotid artery injection of IgG, UMSCs, UMSC-Akt, UMSC-HGF, UMSC-PD-L1, UMSC-PD-L1-Akt, or UMSC-PD-L1-HSF in the post-stroke infarct area.

[0031] Figure 3C The image shows the infarct area after stroke, combined with intravenous and carotid artery injection of IgG, UMSCs, UMSC-Akt, UMSC-PD-L1, or UMSC-PD-L1-Akt.

[0032] Figure 3D The results of post-stroke infarct area are shown in the intravenous (IV), intracarotid (IA), and combined intravenous and intracarotid (IV+IA) injections of UMSC-PD-L1-Akt or UMSC-PD-L1-Akt, and in the control group.

[0033] Figure 3E The results of the infarct area after stroke are shown in the intravenous (IV), intracarotid (IA), and combined intravenous and intracarotid (IV+IA) injections of ADSC-PD-L1-Akt, as well as the control group.

[0034] Figure 3FThe results of intravenous (IV), intracarotid (IA), and combined IV and intracarotid (IV+IA) BMSC-PD-L1-Akt injections and the control group are shown in the following diagrams: infarct area after stroke.

[0035] Figure 4A The results of rats treated with IA-IV-UMSC-PD-L1-Akt, IA-UMSC-PD-L1-Akt, IV-UMSC-PD-L1-Akt, and the control group are shown in the body swing test.

[0036] Figure 4B The results of rats treated with IA-IV-UMSC-PD-L1-Akt, IA-UMSC-PD-L1-Akt, IV-UMSC-PD-L1-Akt, and the control group in the vertical movement test are shown in the figure.

[0037] Figure 4C The results of rats treated with IA-IV-ADSC-PD-L1-Akt, IA-ADSC-PD-L1-Akt, IV-ADSC-PD-L1-Akt, and the control group in the vertical movement test are shown in the figure.

[0038] Figure 4D The results of rats treated with IA-IV-BMSC-PD-L1-Akt, IA-BMSC-PD-L1-Akt, IV-BMSC-PD-L1-Akt, and the control group in the vertical movement test are shown in the figure.

[0039] Figure 4E The diagram shows the neuronal cell death results in rats treated with IA-IV-UMSC-PD-L1-Akt, IA-IV-UMSCAkt, IA-IV-UMSC-PD-L1, and the control group.

[0040] Figure 5A The IVIS results show the biodistribution of UMSC-PD-L1-Akt-Luc injected intravenously, intracarotidally, and combined intravenously and intracarotidally.

[0041] Figure 5B The biodistribution IVIS results at different time points are shown in the intracarotid artery and combined intravenous and intracarotid artery injection of UMSC-PD-L1-Akt-Luc.

[0042] Figure 5C The confocal microscopy immunofluorescence results of combined intravenous and carotid artery injections of UMSC-PD-L1-Akt-Luc are shown.

[0043] Figure 6A-1 and 6A-2A diagram illustrating the gating strategy for analyzing inflammatory cells in the cerebral hemispheres via flow cytometry after IA-IV-UMSC-PD-L1-Akt treatment.

[0044] Figure 6B Demonstrates flow cytometry analysis of CD3 levels in the cerebral hemispheres after IA-IV-UMSC-PD-L1-Akt treatment. + T cells, CD4 + T cells, CD11b + PD-L1 + Results of macrophages and F4 / 80+PD-L1+ microglia.

[0045] Figure 6C Demonstrates flow cytometry analysis of CD11b levels in the cerebral hemispheres after IA-IV-UMSC-PD-L1-Akt treatment. + TNF-α + CD3 + TNF-α + and CD3 + INF-γ + Cellular results diagram.

[0046] Figure 6D-1 A diagram illustrating the gating strategy for analyzing inflammatory cells in the cerebral hemispheres via flow cytometry after IA-IV-UMSC-PD-L1-Akt treatment.

[0047] Figure 6D-2 Demonstrates flow cytometry analysis of CD8 levels in the cerebral hemispheres after IA-IV-UMSC-PD-L1-Akt treatment. + CD8 + CD122 + CD8 + CD122 + IL-10 + and CD19 + IL-10 + Cellular results diagram.

[0048] Figures 7A-1 to 7A-3 A diagram illustrating the gating strategy for spleen cells analyzed by flow cytometry after UMSC-PD-L1-Akt treatment.

[0049] Figure 7A-4 Demonstrating CD11b levels in spleen cells after UMSC-PD-L1-Akt treatment, analyzed by flow cytometry. + PD-L1 + CD11c + PD-L1 + CD19 + PD-L1 + CD4+ PD-L1 + and CD8 + PD-L1 + Cellular results diagram.

[0050] Figure 7B Demonstrating CD3 counts in spleen cells after UMSC-PD-L1-Akt treatment, analyzed by flow cytometry. + TNF-α + CD3 + INF-γ + Cellular results diagram.

[0051] Figure 7C Demonstrating CD8+ in spleen cells after UMSC-PD-L1-Akt treatment, analyzed by flow cytometry. + CD122 + and CD19 + IL-10 + Cellular results diagram.

[0052] Figure 8A The graph shows the infarct area in an AMI model after intravenous (IV), intra-arterial (IA), or a combination of IV and IA injection of UMSC-PD-L1-Akt cells.

[0053] Figure 8B The diagram shows the infarct wall thickness in an AMI model after intravenous (IV), intra-arterial (IA), or a combination of IV and IA (IV+IA) injection of UMSC-PD-L1-Akt or UMSC-PD-L1-HGF cells.

[0054] Figure 8C The results show the infarct area and dead wall thickness in an AMI model after intravenous (IV), intra-arterial (IA), or a combination of IV and IA (IV+IA) injection of ADSC-PD-L1-Akt or UMSC-PD-L1-HGF cells.

[0055] Figure 8D The results show the infarct area and dead wall thickness in an AMI model after intravenous (IV), intra-arterial (IA), or a combination of IV and IA injection of BMSC-PD-L1-Akt cells.

[0056] Figure 9A The graph shows the inflammatory index results 3 days after MI, obtained by intravenous (IV), intra-arterial (IA), or a combination of IV and IA injections of UMSC-Akt-PD-L1.

[0057] Figure 9BThe image shows the results of CD68+ cell infiltration 3 days after MI after intravenous (IV), intra-arterial (IA), or a combination of IV and IA injections of UMSC-Akt-PD-L1.

[0058] Figure 9C This image shows the results of left ventricular fiber analysis on trichrome stained sections 28 days after myocardial infarction, following intravenous (IV), intra-arterial (IA), or a combination of IV and IA injections of UMSC-Akt-PD-L1.

[0059] Figure 10 The IVIS results show the biodistribution at different time points after carotid artery and intravenous injection of UMSC-PD-L1-Akt-Luc. Detailed Implementation

[0060] Unless otherwise defined, all scientific or technical terms used herein have the same meaning as understood by one of ordinary skill in the art to which this invention pertains. Any methods and materials similar to or equivalent to those described herein can be understood and used by one of ordinary skill in the art to practice this invention.

[0061] Unless otherwise stated, all figures used in the specification and claims indicating quantities of ingredients, reaction conditions, etc., should be understood to be modified by the term "about" in all cases. Therefore, unless indicated to the contrary, the numerical parameters specified in the specification and claims of this invention are approximate and may vary according to the desired characteristics sought by the invention. To facilitate understanding of the invention, certain terms are defined below. Further definitions of the following terms, as well as other terms, are consistent throughout this specification. If a definition of a term set forth below differs from the definition in an application or patent incorporated by reference, the definition set forth in this application shall be used to interpret the meaning of that term.

[0062] The term "a" should refer to one or more of the objects of the invention. The term "and / or" refers to one or both of the candidate solutions. The term "a cell" or "the cell" may include multiple cells.

[0063] The term "and / or" is used to refer to two things or either of the two things.

[0064] The term "in vivo" generally refers to something happening inside a living organism. The term "out vivo" generally refers to something happening outside a living organism, such as experiments conducted in an artificial environment created outside of a living organism.

[0065] The terms "genetic engineering" or "genetically engineered cells" refer to the manipulation of genes using genetic material to alter gene copies and / or gene expression levels within cells. The genetic material can be in the form of DNA or RNA. Genetic material can be transferred into cells through various pathways, including viral transduction and non-viral transfection. After genetic engineering, the expression levels of certain genes within a cell can be permanently or temporarily altered.

[0066] The term "transduction" refers to the delivery of genetic material into cells using a virus, which can be an integrating virus or a non-integrating virus. The integrating virus used in this invention can be a lentivirus or a retrovirus. An integrating virus allows its coding gene to integrate into transduced cells infected with viral particles. A non-integrating virus can be an adenovirus or Sendai virus. Non-viral methods can also be used in this disclosure, such as by transfecting DNA or RNA material into cells. The DNA material can be in the form of a PiggyBac, a minicircle vector, or exon plasmids. The RNA material can be in the form of mRNA or miRNA.

[0067] The term "expression vector" refers to a medium that carries foreign genes into cells for expression without degradation. The expression vector in this invention can be a plastid, a viral vector, or an artificial chromosome.

[0068] The term "increased expression" here refers to the increased expression of the RNA or protein of the gene of interest in genetically engineered cells compared to the expression levels of these genes in non-engineered cells.

[0069] Cell “purification” here refers to the isolation and acquisition of cells of interest by utilizing properties specific to those cells. In some embodiments, the unique property refers to the presence or absence of proteins on the cell surface, referred to herein as “surface markers.” In some embodiments, a “positive marker” refers to a surface marker present or expressed on the cells of interest. In some embodiments, a “negative marker” refers to a surface marker not present on the cells of interest.

[0070] The term “treatment” generally refers to achieving the desired pharmacological and / or physiological effect. This effect may be preventative, i.e., completely or partially preventing the disease, disorder, or its symptoms, and may be therapeutic, i.e., partially or completely curing the disease, disorder, and / or the symptoms attributable to it. As used herein, “treatment” covers any treatment of diseases in mammals (preferably humans) and includes (1) suppressing the development of an individual’s disease, disorder, or its symptoms or (2) alleviating or improving an individual’s disease, disorder, or its symptoms.

[0071] The terms “individual,” “subject,” and “patient” used in this article are used interchangeably and refer to any individual mammal that requires diagnosis, treatment, or therapy.

[0072] The term "effective amount" refers to the amount of cells or their derivatives sufficient to produce a beneficial effect on a disease or disorder when administered to a patient or individual requiring treatment. Therapeutic effective amounts will vary depending on the condition and severity of the disease or disorder. It is not limited to the range stated in the specification. Determining a therapeutically effective amount of a given cell or its derivatives is within the scope of ordinary knowledge in the art and does not require further routine experiments.

[0073] The term "pharmaceutical composition" in this invention refers to an effective amount of live cells for the treatment of degenerative diseases. The cellular component may be a mixture of cultured cells or isolated cell populations, such as differentiated tissue cells, progenitor cells, and / or stem cells. The pharmaceutical compositions of this invention are in liquid form or cell suspension buffers, and may contain pharmaceutically acceptable excipients that stabilize the liquid suspension and aid cell survival.

[0074] The term "ischemic condition" as used in this invention refers to a condition caused by or accompanying ischemic disease, typically characterized by reduced blood flow to tissues or organs due to adverse vascular conditions such as vascular stenosis or aneurysm rupture. Myocardial infarction (MI), ischemic stroke, and severe limb ischemia are the three most common ischemic diseases. In some embodiments, the ischemic condition includes acute myocardial infarction (AMI) and ischemic stroke. In one embodiment, the ischemic condition is caused by AMI. In another embodiment, the ischemic condition is caused by ischemic stroke.

[0075] The term "PD-L1" refers to programmed death-ligand 1, a 40 kDa type 1 transmembrane protein encoded by the CD274 gene. PD-L1 binds to its receptor PD-1, which is present on activated T cells, B cells, and bone marrow cells. PD-L1 is also known as "CD274," "B7 homology 1," and "B7-H1."

[0076] Programmed death-ligand 1 (PD-L1), also known as differentiation cluster 274 (CD274) or B7 homology 1 (B7-H1), is a protein encoded by the CD274 gene in humans. Upon binding to its receptor PD-1, PD-L1 inhibits T cell activation, reduces proliferation and cytotoxicity, and induces apoptosis. PD-1 is expressed on the cell surface of activated T cells and B cells. PD-L1 expressed on MSCs interacts with PD-1, providing an inhibitory signal and regulating cell activation and proliferation (J Exp Med 2009; 206: 3015-3029). Furthermore, MSCs have been found to inhibit the proliferation and function of effector T cells through direct contact with activated T cells and indirect secretion of soluble PD-L1 (Stem Cells 2017; 35: 766-776). However, when MSCs are transplanted into the heart to treat infarction, poor transplantation of mesenchymal stem cells has been observed, with little improvement in cardiac function (Ann ThoracSurg 2003;73:1919-1926). Low survival rates of transplanted MSCs have also been found in infarcted hearts, with only 1% viable cells four days after implantation (Circulation 2002;105:93-98).

[0077] Protein kinase B (PKB), also known as Akt, is a serine / threonine-specific protein kinase that plays a crucial role in various cellular processes, including glucose metabolism, apoptosis, cell proliferation, transcription, and cell migration. Akt regulates cell survival and metabolism by binding to and modulating many downstream effector hormones, such as nuclear hormone κB, Bcl-2 family proteins, master lysosomal regulatory hormone TFEB, and mouse dimeric 2 (MDM2). Akt can directly and indirectly promote growth hormone-mediated cell survival. Studies have found that pre-hypoxia conditioning of transplanted cells, i.e., brief culture of cells before transplantation, can protect human brain endothelial cells from ischemic apoptosis by initiating an Akt-dependent pathway (Am J Transl Res. 2017; 9: 664-673). However, there is still room for improvement in the repair and better recovery of Akt-MSCs from ischemic injury.

[0078] This discovery reveals a surprising finding: genetic modification of Akt or HGF and PD-L1 in MSCs not only provides survival signals but also exerts anti-inflammatory effects, significantly improving ischemic conditions. The gene modifications revealed here can maintain and prolong the survival and immunomodulatory capacity of implanted MSCs, overcoming the hypoxic environment of ischemic tissue and the immune system initiated by spleen cells.

[0079] Therefore, this disclosure provides a genetically engineered mesenchymal stem cell population comprising an expression vector containing survival genes (such as Akt or HGF) and immune checkpoint genes (such as PD-L1). It also provides a method for synergistically increasing the survival status and immunomodulatory capacity of MSCs or enhancing MSC proliferation, including transfecting MSCs with Akt or HGF genes and PD-L1 genes, and a method for preventing, improving, and / or treating ischemic conditions, enhancing neurogenesis, or reducing neuronal death, including administering an effective amount of the genetically engineered mesenchymal stem cell population of this disclosure to individuals in need.

[0080] The mesenchymal stem cells disclosed herein can be obtained from various sources, preferably from umbilical cord, adipose tissue, or bone marrow. Depending on the source, mesenchymal stem cells are umbilical cord mesenchymal stem cells (UMSCs), adipose-derived mesenchymal stem cells (ADSCs), and bone marrow mesenchymal stem cells (BMSCs). In some embodiments of this disclosure, MSCs are isolated and purified from the umbilical cord and referred to as “umbilical cord MSCs” or “UMSCs”. In some embodiments, the UMSCs of this disclosure are determined to express the same surface markers and exhibit consistent activity as MSCs isolated from other sources.

[0081] According to this disclosure, mesenchymal stem cells are modified to express programmed death-ligand 1 (PD-L1) and Akt or HGF. As used herein, the term "modified to express" means transferring a foreign gene or gene fragment into mesenchymal stem cells, enabling them to express the foreign gene or gene fragment. Preferably, such modification does not alter the differentiation potential or immunomodulatory properties of the mesenchymal stem cells. In another aspect, such modification is preferably stable, and the expression can be persistent or inducible. The mesenchymal stem cells disclosed are modified to express PD-L1 and Akt or HGF and still possess pluripotent differentiation potential, such as, but not limited to, adipogenesis, chondrogenesis, osteogenic formation, and angiogenesis, similar to ordinary mesenchymal stem cells without PD-L1 and Akt or HGF transduction.

[0082] The method of modifying mesenchymal stem cells with programmed cell death protein-1 (PD-L1) and Akt or HGF is unrestricted. Preferably, PD-L1, Akt, or HGF is transduced using transposons or lentiviruses; more preferably, the transposon is the piggyBac transposon. By applying the piggyBac transposon, PD-L1 and Akt or HGF expression are maintained for 100-150 days. The results indicate that the piggyBac transposon can efficiently and stably transfect bone marrow mesenchymal stem cells, and that gene modification with piggyBac does not alter the DNA copy number and arrangement of the mesenchymal stem cells.

[0083] The genetically engineered mesenchymal stem cells (MSCs) disclosed herein comprise expression vectors containing Akt or HGF genes and PD-L1 genes. In addition to the Akt or HGF and PD-L1 sequences, the vectors disclosed herein also include one or more control sequences to regulate the expression of the polynucleotides disclosed herein. Depending on the expression vector used, manipulating the isolated polynucleotides before inserting them into the vector may be desirable or necessary. Techniques for modifying polynucleotide and nucleic acid sequences using recombinant DNA methods are well known in the art. In some embodiments, control sequences include promoters, leader sequences, polyadenylation sequences, polypeptide sequences, signal peptide sequences, and transcription terminators, etc. In some embodiments, a suitable promoter is selected based on the choice of host cell.

[0084] The recombinant expression vectors disclosed herein, as well as one or more expression regulatory regions, such as promoters and terminators, origins of replication, etc., depending on the type of host they will be introduced into. Non-limiting examples of constitutive promoters include SFFV, CMV, PKG, MDNU3, SV40, Ef1a, UBC, and CAGG.

[0085] In some embodiments, various nucleic acids and control sequences of this invention are linked together to produce a recombinant expression vector, which includes one or more convenient restriction sites to allow the insertion or substitution of the polynucleotides disclosed herein at these sites. Additionally, in some embodiments, the polynucleotides disclosed herein are expressed by inserting the polynucleotides disclosed herein or a nucleic acid construct containing the sequence into a suitable expression vector. In some embodiments involving the creation of expression vectors, the coding sequence is located within the vector so that the coding sequence is operatively linked to a suitable control sequence for expression. The recombinant expression vector can be any suitable vector (e.g., plastid or virus) that facilitates recombinant DNA procedures and yields the expression of the polynucleotides disclosed herein. The choice of vector generally depends on the compatibility of the vector with the host cell into which the vector is introduced. The vector can be a linear or closed circular plastid. In one embodiment, the vector is a viral vector. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors, etc. In some embodiments, the viral vector is a lentiviral vector. Lentiviral vectors are based on or derived from oncoviruses (including MLV-containing retroviral subgroups) and lentiviruses (including HIV-containing retroviral subgroups). Examples include, but are not limited to, human immunodeficiency virus (HIV), equine infectious anaemia virus (EIAV), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV). Additionally, the use of other retroviruses as the vector backbone is also considered, such as murine leukemia virus (MLV).

[0086] In some implementations, the genetically engineered MSCs disclosed herein have been tested in various differentiation assays to establish their conformation to conventional MSCs isolated from other sites in mammals. Differentiation assays included adipogenic differentiation, osteogenic differentiation, and chondrogenic differentiation. In some implementations, differentiation assays also included neuronal cell differentiation.

[0087] This disclosure reveals a genetically engineered MSC population that, by transfecting MSCs with the Akt or HGF gene and the PD-L1 gene, can synergistically improve the survival status and immunomodulatory capacity of MSCs. Therefore, this disclosure provides a method for preventing, improving, and / or treating ischemic conditions, enhancing neurogenesis, or reducing neuronal death, including administering an effective amount of the genetically engineered MSC population disclosed herein to individuals in need.

[0088] In some embodiments, ischemic conditions include, but are not limited to, stroke and myocardial infarction (MI). Preferably, MI is acute myocardial infarction (AMI). Application of the method disclosed herein can attenuate MI-induced fibrosis. Application of the method disclosed herein can also reduce inflammation in ischemic tissue.

[0089] Application can increase the expression of regulatory molecules on individual T cells and reduce inflammatory responses, but it can also enhance CD8 expression in ischemic tissues. + CD122 + Accumulation of Tregs. Compared to unaffected tissue, ischemic tissue treated with the genetically engineered MSCs populations disclosed herein showed an increase in CD3+. + T cells, CD4 + T cells, CD11b + PD-L1 + Macrophages and F4 / 80+PD-L1 + The total number of viable white blood cells, including microglia, increased significantly, while the total number of cells in either the treatment or control group remained unchanged in either tissue.

[0090] This study reveals that an effective amount of genetically engineered MSCs can increase the expression of Akt or HGF and PD-L1. Specifically, the effective amount of the genetically engineered MSCs disclosed in this study ranges from approximately 1 × 10⁻⁶. 5 One to approximately 1 × 10⁹ cells 8 Cells. Other embodiments of this quantity have been disclosed herein.

[0091] The mesenchymal stem cells disclosed herein are contained in injectable formulations. Injectable formulations can be prepared by known methods. For example, injectable formulations can be prepared by, for instance, dissolving, suspending, or emulsifying a pharmaceutical composition in a sterile aqueous or oily medium conventionally used for injection. Examples of aqueous media for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, which can be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (a polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. Examples of oily media include sesame oil, soybean oil, etc., which can be used in combination with solubilizers such as benzyl benzoate, benzyl alcohol, etc. Injectable formulations prepared in this way are preferably packaged in suitable ampoules.

[0092] The route of administration of MSCs in this invention depends on the tissue or organ requiring treatment. In the case of individuals with myocardial infarction, the route of administration of MSCs may be intravenous injection, arterial injection, or a combination thereof. The cell-containing solution can be prepared using suitable diluents such as water, ethanol, glycerol, liquid polyethylene glycol, various oils and / or mixtures thereof, and other diluents known to those skilled in the art. In one particular embodiment, in individuals with stroke or AMI, administration is a combination of intracarotid and intravenous injection. In another particular embodiment, the effective amount of this administration ranges from approximately 1 × 10⁻⁶ for intracarotid injection. 5 One cell to approximately 3 × 10 6 10 cells and approximately 3 × 10 intravenously injected 5 One cell to approximately 3 × 10 6 Each cell.

[0093] The administration according to this disclosure also includes injecting mesenchymal stem cells into the individual requiring this treatment via an intra-arterial route combined with a venous route. In a preferred embodiment of this disclosure, the intra-arterial route is via the carotid artery.

[0094] In some embodiments of the present invention, the process of administering MSCs is referred to as “transplantation” or “implantation”.

[0095] In one embodiment, the engineered stem cells disclosed herein may be administered together with an additional activator. In some embodiments, the engineered stem cells and the additional activator may be administered simultaneously, separately, or concurrently. In one embodiment, the engineered stem cells and the additional activator may be administered periodically.

[0096] It should be understood that any reference to any prior art publication herein does not constitute an admission that such publication constitutes part of the general knowledge in this field.

[0097] While some details have been disclosed through illustration and examples for clarity of understanding, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit or scope of the disclosure. Therefore, the foregoing descriptions and examples should not be construed as restrictive.

[0098] Example

[0099] Methods and materials:

[0100] Preparation, isolation, and identification of umbilical cord mesenchymal stem cells (UMSCs), umbilical cord adipose-derived mesenchymal stem cells (ADSCs), or bone marrow mesenchymal stem cells (BMSCs)

[0101] Human umbilical cord tissue was collected and washed three times with calcium- and magnesium-free PBS (DPBS, Life Technology). The umbilical artery, vein, and adventitia were dissected along the midline using scissors to separate from Wharton's jelly (WJ). The contents of the Wharton's jelly were then extensively cut into pieces smaller than 0.5 cm³, treated with collagenase type 1 (Sigma, St. Louis, USA), and cultured at 37°C in 95% air / 5% CO2 humidified air for 3 hours. The explants were then cultured in DMEM containing 10% fetal bovine serum (FCS) and antibiotics at 37°C in 95% air / 5% CO2 humidified air. They were then kept undisturbed for 5–7 days to allow cell migration from the explants. The cell morphology of umbilical cord mesenchymal stem cells (UMSCs) became uniformly spindle-shaped after 4–8 passages, and cell surface molecules from WJ were characterized by flow cytometry analysis. Cells were separated in PBS with 2 mM EDTA, washed with PBS (Sigma, USA) containing 2% BSA and 0.1% sodium azide, and co-cultured with corresponding antibodies conjugated to fluorescein isothiocyanate (FITC) or phycoerythrin (PE), including CD13, CD29, CD44, CD73, CD90, CD105, CD166, CD49b, CD1q, CD3, CD10, CD14, CD31, CD34, CD45, CD49d, CD56, CD117, HLA-ABC, and HLA-DR (BD, PharMingen). Cells were then analyzed using a Becton-Dickinson flow cytometer (Becton Dickinson, San Jose, CA). Adipose-derived mesenchymal stem cells (ADSCs) and bone marrow mesenchymal stem cells (BMSCs) were purchased from ATCC (ADSCs, PCS 500-011; BMSCs, PCS 500-012).

[0102] Plast construction

[0103] Akt plasmid (0.1 μg) (pCMV6-myc-DDK-Akt, OriGene), HGF (0.1 μg) (pCMV6-XL4-HGF, OriGene), and PD-L1 plasmid (0.1 µg) (pCMV6-myc-DDK-PD-L1, OriGene) containing Akt, PD-L1, and GFP cDNA were transferred into pIRES (Clontech) or pSF-CMV-SbfI (Oxford Genetics) using specific restriction enzyme ligands (EcoR1 and Nhe1 in TK, BamH1 and Not1 in PD-1) to construct pSF-PD-L1-Akt, pSF-PD-L1-HGF, pSF-Akt-GFP, and pSF-PD-L1-GFP constructs, etc., according to the manufacturer's instructions. DNA (Roche) was transfected into UMSCs, ADSCs, or BMSCs to obtain UMSC-PD-L1-Akt, UMSC-PD-L1-HGF, UMSC-Akt-GFP, UMSC-HGF-GFP, and UMSC-PD-L1-GFP.

[0104] Lentiviral plasmids

[0105] Slow vector (pLAS3w) and packaging (psPAX2) / encapsulated plasmid (pMD2.G) were obtained. cDNA encoding full-length human Akt, PD-L1, luciferase (Luc), and control GFP were obtained through recombination with pCMV6-myc-DDK-Akt, pCMV6-XL4-HGF, pCMV6-myc-DDK-PD-L1, and pRMT-Luc (OriGene). Constructs pUltra-PD-L1-Akt, pUltra-PD-L1-HGF, pUltra-Akt-GFP, pUltra-HGF-GFP, and pUltra-PD-L1-GFP were obtained by specifically introducing pUltra (Addgene) or pSF-CMV-CMV-Sbf1 (Oxford Genetics) into these constructs. Subsequently, these templates were amplified by PCR using specific primers, and then replicated to the lentiviral vector backbone pLAS3w using restriction endonucleases. To prepare recombinant lentiviruses carrying PD-L1-Akt, Akt, PD-L1, Luc, and control GFP, the recombinant lentiviruses were transfected into 293T cells at a ratio of 3:3:1 (XtremeGene HP DNA (Roche) transfection). After 36 hours, the culture supernatant containing viral particles was collected, and again at half volume after 24 hours. The supernatant was then centrifuged at 15,000 rpm for 10 min to remove debris, and transferred to 36 mL ultracentrifuge tubes. The supernatant was then ultracentrifuged at 25°C for 25,000 rpm for 3 hours. The lentiviral particles were resuspended. The virus was thawed immediately before pricing and cell transduction. Bone marrow mesenchymal stem cells were infected with a suitable lentivirus, achieving a gene transfer efficiency of at least 80%.

[0106] Lentiviral transduction

[0107] Lentiviral plasmid transduction was performed in 6-well plates. Unless otherwise specified, UMSCs were seeded at 1 × 10⁵ cells per well in triplicate, resulting in a final volume of 1 ml / well and a fold increase in infection (MOI) of 5. A stock solution of 5 mg / ml protamine sulfate (Sigma-Aldrich) (sterile filtered in DMEM-LG) was added to obtain the desired final concentration. Cells were transduced for 24 hours, then replaced with 1.5 ml / well to obtain UMSC-PD-L1-Akt, UMSC-Akt-GFP, UMSC-Luc, and UMSC-PD-L1-GFP. Overgrown cells were seeded into 6-well plates for drug selection using 1.0 mg / ml G418 or puromycin solution (Sigma). The medium was changed every 2 days. Green fluorescent protein (GFP) expression was observed using an inverted fluorescence microscope based on the color of the medium and cell status. After 7 days of selection, the medium was replaced with intact medium without puromycin, and culture continued.

[0108] Construction of the stable cell line piggyBac transposon system

[0109] The piggyBac vector from pPB-CMV-MCS-EF1α-RedPuro (System Bioscience) was used as the basic vector, containing multiple replication sites (MCS), piggyBac terminal repeats (PB-TRs), core insulators (CIs), and a puromycin selection hormone (BSD) fused to the RFP, driven by human EF1α. PCR amplification was performed on DNA fragments containing PD-L1-Akt, PD-L1-HGF, Akt, HGF, or PD-L1 (from pUltra-PD-L1-Akt, pUltra-PD-L1-HGF, pUltra-Akt, pUltra-HGF, pUltra-PD-L1), which were subdivided into the pPB-CMV-MCS-EF1α-RedPuro vector, located before the EF1α coding region. Detailed information regarding vector construction (pPB-PD-L1-Akt, pPB-PD-L1-HGF, pPB-Akt, pPB-HGF, pPB-PD-L1) is available upon request. To obtain stable UMSCs cells, the above-mentioned pPB-PD-L1-Akt, pPB-PD-L1-HGF, pPB-Akt, pPB-HGF, and pPB-PD-L1 vectors were co-transfected into UMSCs, ADSCs, and BMSCs cells via calcium phosphate (Invitrogen) or electroporation (Amaxa nucleafector II, Lonza) with a piggyBac transfection expression vector (System Biosciences). Stable cells were selected in the presence of puromycin.

[0110] In vitro proliferation, migration and differentiation assays

[0111] To detect cell proliferation and migration, UMSC-PD-L1Akt and UMSCs were compared using CFSE staining and transwell migration assays. CFSE staining was applied to detect the proliferation of UMSC-Akt-PD-L1 or UMSCs and to evaluate its effect on cell proliferation. CFSE-stained UMSC-Akt-PD-L1 cells were analyzed at a concentration of 2 × 10⁻⁶ cells / well. 4 Cells were seeded at a density of 100 cells / mL and incubated at 37°C and 5% CO2 for 5 days. The general gating strategy for proliferating cells was the same as that for PBMCs. Fluorescence measured in this proliferation gating corresponds to proliferative activity during the 5-day experiment. All data were analyzed using FlowJo 8.7 software after acquisition.

[0112] Cell migration assays were performed according to the manufacturer's instructions (Costar, #3421). 100 μL of UMSC-Akt-PD-L1 or UMSCs were placed in the upper chamber (transwell: 6.5 mm diameter, 5.0 mm pore size). SDF-1α (100 ng / mL, R&D System, positive control) was used in the lower chamber. Cells were incubated at 37°C for 4 hours in a 5% CO2 incubator. Since almost all cells remained below the membrane after migration, quantification was achieved simply by counting these cells. As before, the adherent cells below the membrane were counted under a microscope.

[0113] Adipogenic differentiation was induced by culturing fusion monolayers of UMSC-Akt-PD-L1 or UMSCs in adipogenic differentiation medium containing DMEM high glucose (DMEM-HG, Sigma), 100 U / mL penicillin, 100 mg / mL streptomycin, 100 mM insulin (Sigma), 500 mM 3-isobutyl-1-methylxanthine (Sigma), 1 mM dexamethasone (Sigma), 100 mM indomethacin (Sigma), and 10% FCS. Cells preserved in standard UMSCs medium were used as a negative control. The adipogenic differentiation medium was changed three times a week. To assess adipogenic differentiation, cells were stained with 0.3% Oil Red O (Sigma) for 10 minutes at room temperature (to mark intracellular lipid accumulation) and counterstained with hematoxylin.

[0114] To induce osteogenic differentiation, fused monolayers of UMSC-Akt-PD-L1 or UMSCs were cultured in DMEM high-glucose (DMEM-HG, Sigma) containing 100 U / mL penicillin (Sigma), 100 mg / mL streptomycin (Sigma), 50 mg / mL L-ascorbic acid 2-phosphate (Sigma), 10 mM β-glycerophosphate (Sigma), 100 nM dexamethasone (Sigma), and 10% FCS. Ordinary UMSCs medium served as a negative control. The osteogenic differentiation medium was changed three times weekly. Osteogenic levels were determined using Alizarin Red S staining (1%, Sigma) to detect calcium mineralization.

[0115] Chondrogenic differentiation of UMSC-Akt-PD-L1 or UMSC cells was induced using a high-density granulocyte culture system. Cells were washed in serum-free chondrogenic differentiation medium containing DMEM-HG, 100 U / mL penicillin, 100 mg / mL streptomycin, 50 mg / mL L-ascorbic acid 2-phosphate, 40 mg / mL proline (Sigma), 100 mg / mL sodium pyruvate (Sigma), 100 nM dexamethasone, and ITS adducts (10 mg / mL bovine insulin, 5.5 mg / mL transferrin, 5 mg / mL sodium selenite, 4.7 mg / mL linoleic acid, and 0.5 mg / mL bovine serum albumin (Sigma)). Aliquots of 250,000 cells were resuspended in chondrogenic differentiation medium, centrifuged at 250 × g, and then 10 ng / mL TGF-β1 (R&D Systems) was added. Chondrogenic differentiation medium without TGF-β1 served as a negative control. The medium was changed twice weekly. Histological confirmation of chondrogenic differentiation in granular cultures was achieved by Alsin Blue staining (Sigma) with sulfated proteoglycans. Furthermore, UMSC-Akt-PD-L1 or UMSCs were cultured in EBM (Cambrex) for 2–3 days, followed by culturing in 24-well plates pre-coated with matrix gel (300 μL / well; Becton-Dickinson) and vascular endothelial growth hormone (VEGF, 10 ng / ml, Sigma) for 2–3 days to induce endothelial cell differentiation into blood vessel formation.

[0116] To induce neural cell differentiation, UMSC-Akt-PD-L1 or UMSCs were co-incubated with DMEM using a three-step method. In short, during the neural induction step, cells were low-density coated onto 6-well plates containing fibronectin (Sigma) and then sequentially exposed to (1) DMEM-HG (Sigma) containing 10% FCS and 10 ng / mL bFGF (R&D Systems) for 24 hours; (2) during the neural commitment step, DMEM-HG containing 1 mM β-mercaptoethanol (βME, Sigma) and 10 ng / mL NT-3 (R&D Systems) for 2 days; and (3) during the neural differentiation phase, DMEM-HG containing NT-3 (10 ng / mL, R&D Systems), NGF (10 ng / mL, R&D Systems), and BDNF (50 ng / mL, R&D Systems) for 3–7 days. After differentiation, cells were reserved for immunohistochemical analysis.

[0117] Flow cytometry

[0118] To analyze the expression of cell surface markers, cells were isolated in PBS with 2 mM EDTA, washed with PBS containing BSA (2%) and sodium azide (0.1%), and then incubated with antibodies conjugated to fluorescein isothiocyanate (FITC) or phycoerythrin (PE), respectively, until analysis. As a control, staining with mouse IgG1 isotype control antibodies was performed. Antibodies against PD-1, PD-L1, CD3, CD8, CD4, CD25, Foxp3, CD44, CD45, CD11b, F4 / 80, IFN-γ, CD206, and GFP used for flow cytometry were purchased from BD Biosciences. Cell analysis was performed using FACScan (BD) and CellQuest Analysis (BD Biosciences) and FlowJo v.8.8 software (TreeStar). Results are expressed as the percentage of positive cells out of the total number of cells. To quantitatively compare the expression of surface proteins, the fluorescence intensity of each sample was expressed as median fluorescence intensity (MFI). For intracellular staining of Ki-67 and granzyme B, TILs were cultured for 48 hours in the presence of 1 μg / ml anti-CD3. Cells were then cultured with anti-CD8 before Triton x100 infiltration and stained with anti-Ki-67 antibody (Millipore) and Granzyme B. Data were analyzed using FACScan (BD), CellQuest Analysis (BD Biosciences), and FlowJo v.8.8 (TreeStar).

[0119] hypoxia program

[0120] UMSC-Akt-PD-L1 or UMSC (1×10⁵ / mL) were cultured at 37°C in a humidified incubator with 5% CO₂, and treated at different time points under normoxic (21% O₂) or hypoxic (1% O₂) conditions. Hypoxic culture was performed in a dual-gas incubator (Jouan, Winchester, Virginia) equipped with an O₂ probe to regulate N₂ levels. Cell number and viability were evaluated using the trypan blue rejection assay and the TUNEL assay.

[0121] Assay for hydrogen peroxide-induced cell death

[0122] Cell viability was assessed using the MTT assay (C, Ndipheyl-N-4,5-dimethylthiazolyl-2-yltetrazole ammonium bromide, Sigma). UMSCs were cultured in 96-well plates. After incubation with hydrogen peroxide for 1 h, the medium was replaced with MTT solution (5 mg / mL in PBS). Incubation continued for 4 h, followed by aspiration to remove the supernatant. Dimethyl sulfoxide (DMSO, Sigma) was added, and absorbance was read at 570 nm using a molecular device to determine the percentage of cell viability. Furthermore, the nonpolar compound DCFH-DA (Sigma) entered the cells, was cleaved to form DCFH, and was then captured and oxidized by oxygen free radicals to generate fluorescent DCF. UMSCs were pre-cultured in serum-free DMEM for 24 h, treated with H2O2 for 30 min, and pre-loaded with 10 µM DCFH-DA at 37 °C for 30 min. Fluorescence intensity was analyzed using a Finland fluorescence reader with a 485 nm excitation and 538 nm emission filter.

[0123] In vitro analysis of antigen-specific T cell responses

[0124] BALB / c mouse spleen cells (2×10) 6 Place the cells on RPMI-1640 medium (Gibco) in a 24-well plate, add 10% FBS (Sigma) and 1% penicillin / streptomycin (Gibco). Then, add cells (2×10⁶ cells / wells) at different ratios (10:1 or 1:1). 5 Or 2×10 6Spleen cells were co-cultured either unstimulated or with CD3-CD28 microbeads (dynabeals, thermal). For proliferation assays, spleen cells were stained with carboxyfluorescein succinimide (CFSE) (Invitrogen). The proliferation / division ratio was estimated using the proliferation index (PI), calculated as: PI = total number after proliferation / total number before proliferation. Cells were harvested after 6 days of culture for staining to analyze the proliferation of Treg, CD4, and CD8-T cell subsets. Alternatively, to analyze proliferation in longitudinal samples after 6 days of culture with limited cell numbers, unstained spleen cells were cultured and stained with Ki67 or isotype control antibodies. The fold change (FC proliferation) was calculated as the proliferation rate under UMSC-TRAIL-TK-PD-1 conditions divided by the proliferation rate under control conditions.

[0125] TUNEL Analysis

[0126] Apoptosis was detected using immunohistochemistry with a commercial TUNEL staining kit (DeadEnd fluoremic TUNEL system; Promega). The percentage of TUNEL-labeled cells was expressed as the number of TUNEL-positive nuclei divided by the total number of DAPI-stained nuclei.

[0127] Animal brain ischemia-reperfusion model

[0128] Adult male Sprague-Dawley rats (weighing 250-300g) were selected. Animals underwent three-vessel ligation. All surgical procedures, animal experimental protocols, and methods were performed according to institutional guidelines. Rats were anesthetized with chloral hydrate (0.4g / kg, ip), and the right middle cerebral artery (MCA) and bilateral common carotid arteries (CCAs) were ligated. The bilateral CCAs were clamped with non-traumatic arterial clips. A 2×2 mm craniotomy was drilled at the fusion of the zygomatic bone and squamous bone using a surgical microscope. The right middle cerebral artery was ligated with L0-0 nylon suture. Cortical blood flow in the anesthetized animals was continuously measured using a laser Doppler flowmeter (PF-5010, Periflux system, Sweden). A 1 mm diameter puncture was created in the right frontoparietal region to place a photodetector. The probe (0.45 mm diameter) was stereotactically positioned in the cortex (1.3 mm posteriorly, 2.8 mm lateral to the fontanelle, and 1.0 mm subdurally). After 90 minutes of ischemia, the sutures on the MCA and the arterial clamp on the CCA were removed to allow reperfusion. Core body temperature was monitored with a thermistor probe and maintained at 37°C using a heating pad during anesthesia. After recovery from anesthesia, body temperature was maintained at 37°C using a heating lamp.

[0129] Carotid artery and / or vein transplantation of UMSC-PD-L1-Akt, UMSC-PD-L1-HGF, ADSC-PD-L1-Akt or BMSC-PD-L1-Akt

[0130] Two hours after cerebral ischemia, adult male Sprague-Dawley rats (200-250 g) were anesthetized with chloral hydrate (0.4 g / kg, ip) and injected intravenously with approximately 1 × 10⁻⁶ ppm. 6 Cells (UMSC-PD-L1-Akt, UMSC-PD-L1-HGF, UMSC-Akt, UMSC-HGF, or UMSC-PD-L1). Control group animals received only PBS. During intracarotid artery injection, the ipsilateral common carotid artery was re-exposed, and the external carotid artery was ligated with 6-0 silk to induce coagulation of the superior thyroid artery and pterygopalatine artery. 24 hours post-stroke, 1×10⁻⁶ cells were injected using a 24-G catheter. 5 UMSC-PD-L1-Akt, UMSC-PD-L1-HGF, UMSC-Akt, UMSC-HGF, UMSC-PD-L1, ADSC-PD-L1-Akt, or BMSC-PD-L1-Akt were injected into the internal carotid artery. Because mesenchymal stem cells possess immunosuppressive properties, the rat host did not receive any immunosuppressive drugs.

[0131] Neurobehavioral assessment

[0132] Behavioral assessments were performed 5 days before cerebral ischemia and 1, 7, 14, and 28 days after cell transplantation. Tests measured body asymmetry, motor function, and grip strength. Baseline test scores were recorded to standardize the scores after cerebral ischemia. A body-swing test was used to assess body asymmetry after middle cerebral artery ligation. Initially, animals were examined for lateral movements with their bodies suspended by their tails 10 cm above the cage floor. The initial head-swing frequency on the ischemic side was calculated over 20 consecutive tests and standardized to baseline scores. Motor activity was measured using a VersaMax animal activity monitor (Accuscan Instruments, Inc., Columbus, OH) for approximately 2 hours of behavioral recording. This instrument contained 16 horizontal and 8 vertical infrared sensors. The vertical sensors were located 10 cm above the chamber floor. Motor activity was calculated as the number of beams disrupted by a mouse's movement within the chamber. Three parameters of the vertical items were calculated for more than 2 hours: (i) vertical activity, (ii) vertical time, and (iii) number of vertical movements.

[0133] Measuring infarct area using magnetic resonance imaging (MRI)

[0134] MRI was performed on anesthetized rats using a 3.0T R4 imaging system (GE). The brain was scanned using 6–8 coronal slices, each 2 mm thick and seamless. Spin-echo imaging was employed (repetition time 4000 ms; echo time 10 ms). 5 T2-weighted imaging (T2WI) pulse sequences were acquired in milliseconds. Images were acquired in each animal at 1, 7, and 28 days post-cerebral ischemia. To measure the infarct area in the right cortex, the non-infarcted area of ​​the right cortex was subtracted from the total cortical area in the left hemisphere. The infarct area was plotted manually layer by layer, and the volume was calculated using internal volume analysis software (Voxtool, General Electric).

[0135] Bioluminescence imaging

[0136] Animals were imaged using the IVIS Imaging System 200 (Xenogen) to record bioluminescent signals. Animals were anesthetized with isoflurane and intraperitoneally injected with D-luciferin (Caliper) at a dose of 270 mg / g body weight. Images were acquired 15 minutes after luciferin injection. For BLI analysis, the IVIS system (Xenogen) was used to identify regions of interest, including intracranial signal areas, and total photon flux was recorded. To facilitate comparison of cell engraftment rates, each animal's luminescence score was standardized on day 14 based on its own luminescence readings, thus serving as a control for each mouse.

[0137] White blood cells were isolated from the spleen and brain.

[0138] Spleens were harvested from individual rats in both the control and treatment groups, and single-cell suspensions were prepared by passing the tissue through a 100 μm nylon mesh (Fisher Scientific). Cells were washed with RPMI 1640 (Invitrogen). Red blood cells were lysed with 1× erythrocyte lysis buffer (eBioscience) and incubated for 3 min. Cells were then washed twice with RPMI 1640, counted, and resuspended in stimulation medium containing 2% fetal bovine serum (Gibco) and 0.4% β-ME (Sigma-Aldrich).

[0139] The brain was divided into ischemic (right) and non-ischemic (left) hemispheres and digested with 3 U / mL recombinant DNase I (Roche) and 1 mg / mL Clostridium histolyticum collagenase (Sigma-Aldrich). The digested cells were resuspended in 80% Percoll (GE Healthcare) and centrifuged at 1600 rpm (400 g) for 30 minutes using a density gradient. Eighteen leukocytes were extracted from the interphase, washed twice with RPMI 1640, counted, and resuspended in stimulation medium. Inflammatory cells in individual cerebral hemispheres were detected using flow cytometry.

[0140] Flow cytometry detection of T cell populations

[0141] Cell populations were analyzed using multicolor flow cytometry. First, cells were washed with PBS containing BSA (2%) and sodium azide (0.1%). Then, prior to analysis, cells were cultured with the corresponding antibodies from BD (PD-L1, CD3, CD8, CD4, CD25, Foxp3, CD44, CD45, CD11b, F4 / 80, IFN-γ, 7AAD, and CD206) before analysis.

[0142] Mouse IgG1 isotype control antibody staining was used as a control. FACScan (BD) and CellQuestAnalysis (BD Biosciences) and FlowJo software v.8.8 (TreeStar Inc.) were used. The gating strategy was based on correct identification of the first gate, exclusion of duplicates by SSC-A and SSC-H, exclusion of dead cells, and further selection for 7-AAD+ / FSC-A. Results are expressed as the percentage of positive cells out of the total number of cells. Differences between the two groups were analyzed using Newman-Keuls post-hoc test. A p-value <0.05 was considered significant.

[0143] Intracellular staining

[0144] Intracellular staining was performed. Briefly, isolated leukocytes (2 × 10⁶ cells / mL) were resuspended and cultured for 4 hours with LPS (10 μg / mL, Sigma), phorbol 12-myristate 13-acetate (50 ng / mL, Sigma), iomycin (500 ng / mL), and GolgiPlug protein transport inhibitor (BD Biosciences). Cells were fixed and permeated with fixation / permeation buffer (BD Biosciences) according to the manufacturer's instructions. Cells were stained with tumor necrosis hormone-α (replication MP6-XT22), interleukin (IL)-10 (replication JES5-16E3), PD-1 (replication J43), and FoxP3 (replication FJK-16s) and then resuspended in staining buffer for collection. Isotype-matched single replication antibodies were used as negative controls.

[0145] Cytokine assay

[0146] To detect the levels of TNF-α, VEGF, and TGF-β, tumor-infiltrating lymphocytes (TILs) were isolated from mice treated with different doses of IO@FuDex formulation 4 weeks after tumor inoculation. The TILs were cultured in PRMI-1640 medium and L-glutamine (2 mM) in 6-well discs (2 × 10⁻⁶ m²). 5 cells / ml -1TILs were further cultured in the medium for 48 h. TNF-α, TGF-β, and VEGF in the culture medium were semi-quantitatively measured using the Quantikine ELISA kit (R&D Systems) under a spectrophotometer (Molecular Devices), and standard curves were generated using the SOFTmax (Molecular Devices) program.

[0147] Immunohistochemical assessment

[0148] Animals were anesthetized with chloral hydrate (0.4 g / kg, ip), and their brains were fixed by cardiac perfusion with physiological saline, followed by perfusion with 4% PFA. Tissue samples were then collected, further fixed by immersion in 4% PFA, dehydrated in 30% sucrose, and frozen on dry ice. Coronal sections (6 μm thick) were cut using a cryostat, stained with H&E, and observed under an optical microscope (E600, Nikon). The expression of luciferase + cell type-specific markers in cells was detected using a dual immunofluorescence assay. Each coronal section was first stained with primary luciferase antibody (1:1000, Novus), MAP-2 (1:200, Millipore), GFAP (1:500, Millipore), and GFP antibody (1:200, Millipore), and then double immunostained with specific antibodies to Cy3 (1:500; goat anti-rabbit IgG, Jackson Immunology Research) or FITC (1:500; goat anti-mouse IgG, Jackson Immunology Research) bound to a secondary antibody to demonstrate their colocalization in a cell under CLSM.

[0149] Assessment of immune-related adverse events (irAEs)

[0150] Following treatment with IO@FuDex3 and M-IO@FuDex3, irAEs were assessed, including: (1) body weight monitoring, (2) histology, (3) immune cell infiltration, and (4) liver and kidney function. Mouse body weight was monitored during treatment. Additionally, at 4 weeks post-tumor inoculation (n=6), liver, lung, spleen, kidney, and colon sections from mice treated with the IO@FuDex formulation were stained with H&E and subjected to histological analysis. CD8+ and CD4+ T cell infiltration in the liver, colon, kidney, and lung were examined by IHC, and the number of positive cells per 10 high-power fields per square millimeter was calculated for scoring. Furthermore, biochemical characteristics of ALT, AST, creatinine, and glucose were determined using mouse serum at consecutive time points (0, 5, 10, 15, 20, 25, and 30 days) in each group (n=6) using a Beckman Unicell DxC800 analyzer.

[0151] Rat model of acute myocardial infarction and treatment regimen

[0152] Adult male Sprague-Dawley rats (SD, 200–250 g) were induced with acute myocardial infarction (AMI) by ligation of the left anterior descending coronary artery (LAD). Briefly, after anesthesia with 2% isoflurane in 100% oxygen, the rats were mechanically ventilated using a ventilator (SN-480-7, Japan) at an expiratory volume of 1 mL / 100 g and a respiratory rate of 80 / min. A left thoracotomy was performed at the 4th–5th intercostal space using a rib retractor (MY-9454S, Japan); the left lung was deflated with a small piece of saline-soaked gauze. The pericardium was then removed, and intramyocardial ligation was performed 1–2 mm below the atrioventricular groove using a 6-O polyethylene suture needle (Ethicon, UK). The pleural cavity was then re-inflated before closure. The sham-operated group rats received the same treatment protocol except for coronary artery ligation.

[0153] AK-PD-L1-PD, UMSC-PD-L1-HGF, AK-PD-L1-Akt, or BMTID-L1-PD transplantation

[0154] Two hours after acute myocardial infarction, rats were anesthetized with chloral hydrate (0.4 g / kg, ip) and intravenously injected with 1×10 6 Cells (UMSC-PD-L1-Akt, UMSC-PD-L1-HGF). Control group animals were given PBS only. During intracarotid artery injection, the left common carotid artery was exposed, the external carotid artery was ligated with 6-0 silk, and the superior thyroid artery and pterygopalatine artery were coagulated. 24 hours after acute myocardial infarction, 0.5 × 10⁻⁶ cells were injected via a 30g vascular catheter. 6 UMSC-Akt-PD-L1, UMSC-PD-L1-HGF, ADSC-PD-L1-Akt, or BMSC-PD-L1-Akt are injected into the internal carotid artery.

[0155] Myocardial histology and immunohistochemical analysis

[0156] Experimental rats were re-anesthetized with chloral hydrate (0.4 g / kg IP) and sacrificed 3 and 28 days after acute myocardial infarction (AMI), respectively. Three rats without ligation of the left ventricular artery (LAD) served as normal controls. The rat hearts were fixed in 4% paraformaldehyde and immersed in 30% sucrose for 3 days. A series of 6 μm thick sections were cut from each tissue block in the coronal plane using a cryostat, stained with H&E, and analyzed using an optical microscope (Nikon, E600). Masson trichrome (Sigma) stained sections at different levels along the long axis (apex, mid-left ventricle, and base) were analyzed using ImageJ software (NIH) to calculate infarct area, wall thickness, and fibrosis percentage. Infarct area was measured as a percentage of LV circumference in Masson trichrome stained sections 28 days after myocardial infarction. The grading of inflammation and fibrosis was performed blinded using a scoring system under an optical microscope: Grade 0, no inflammation or fibrosis; Grade 1, up to 5% of cardiac segments have cardiac infiltration or fibrosis; Grade 2, 6%-10%; Grade 3, 11%-30%; Grade 4, 31%-50%; Grade 5, >50%.

[0157] Ten high-power fields were randomly selected from the marginal zone of the infarcted myocardium, and inflammatory cell infiltration (CD68 positive) was detected by primary CD68 staining (1:200, milipore). The number of cells per high-power field was expressed as the number of cells per field. Tissue sections were analyzed using a Carl Zeiss LSM510 laser scanning confocal microscope. Immunofluorescence-labeled slides were excited at 488 nm, 543 nm, and 680 nm using FITC (green, 1:500; Jackson Immunoresearch), Cy3 (red, 1:500; Jackson Immunoresearch), and Alexa Fluor 680 (blue, 1:1000; Invitrogen), respectively.

[0158] Statistical analysis

[0159] All measurements were performed in a blinded design. Results are expressed as mean ± SEM. The significance of the mean difference between the control and treatment groups was assessed using a two-tailed Student's t-test. Differences between the two groups were analyzed using Newman-Keuls post-hoc tests. A p-value <0.05 was considered significant.

[0160] Example 1: In vitro properties of UMSCs and UMSC-PD-L1-Akt

[0161] Primary cultures of umbilical cord mesenchymal stem cells (UMSCs) were prepared from Wharton jelly (WJ), and their cell morphology and biological characteristics were analyzed. Figure 1AFlow cytometry showed that CD1q, CD3, CD10, CD14, CD31, CD34, CD45, CD49d, CD56, CD117, and HLA-DR were negative, but CD13, CD29, CD44, CD73, CD90, CD105, CD166, CD49b, and HLA-ABC were positive. Figure 1B These observations indicate that UMSCs possess the same surface markers as mesenchymal stem cells (MSCs), consistent with findings observed in bone marrow mesenchymal stem cells.

[0162] Flow cytometry was used to detect RFP fluorescence and PD-L1 expression levels in UMSC-PD-L1-Akt. From 36 to 48 hours post-transfection, flow cytometry results for RFP and PD-L1 showed an average uptake efficiency of 55-65%. Figure 1C Subsequently, after 3-5 days of puromycin selection, over 90% of the cells were completely transformed by the transgene. Figure 1C In the Western ink dot method, a significant increase in Akt transgene expression was also observed in UMSC-PD-L1-Akt. Figure 1D ).

[0163] To determine the in vivo survival rate of UMSC-PD-L1-Akt-Luc, the number of UMSCs expressing Luc signal intensity in vivo was observed to be directly correlated with the in vitro rate within the experimental range. UMSC-PD-L1-Akt-Luc maintained luciferase expression for 100-150 days. Figure 1D These results demonstrate that the piggyBac transposon can efficiently and stably transfect bone marrow mesenchymal stem cells, and that transposonin gene modification does not alter the DNA copy number or arrangement of mesenchymal stem cells.

[0164] To demonstrate whether UMSC-PD-L1-Akt still possesses pluripotent differentiation potential, adipogenesis, chondrogenesis, osteoogenesis, and angiogenesis were analyzed. The results showed that UMSC-PD-L1-Akt exhibited similarity to ordinary UMSCs not transduced with PD-L1 and Akt. Figure 1E ).

[0165] Example 2: Enhancing UMSC-PD-L1-Akt proliferation

[0166] To demonstrate the proliferative effect, the biological characteristics of UMSC-PD-L1-Akt were examined using CFSE analysis. Compared to UMSC, a significant increase in proliferation was observed in UMSC-PD-L1-Akt, with the percentage of proliferating cells observed in CFSE analysis compared to UMSC, without affecting cell viability. Figure 2A ).

[0167] Example 3: Enhancing UMSC-PD-L1-Akt survival rate in H2O2-induced apoptosis

[0168] To investigate the mechanism by which H2O2 promotes cell survival, we observed the effect of H2O2 on apoptosis in UMSC-PD-L1-Akt cells in vitro. Compared with UMSC-Luc and control UMSCs, UMSC-PD-L1-Akt cells showed greater resistance to H2O2-induced cell death in a dose-dependent manner (0, 1, 10, 100 µM). Figure 2B Compared to UMSCs, TUNEL in UMSC-PD-L1-Akt was reduced after administration of hydrogen peroxide. + The number of cells was significantly reduced ( Figure 2C After H2O2 treatment, intracellular reactive oxygen species (ROS) levels analyzed by DCF fluorescence showed that UMSC-PD-L1-Akt was significantly reduced compared to UMSCs. Figure 2D In Western ink spot analysis, Akt levels in UMSCs and UMSC-PD-L1-Akt were induced to phosphorylate within 30 minutes after H2O2. Figure 2E Importantly, phosphorylated Akt levels recovered to near baseline in UMSCs, but remained significantly elevated in UMSC-PD-L1-Akt after 3 hours. Figure 2E ).

[0169] Example 4: Infarct volume reduction after combined intravenous injection of UMSC-PD-L1-Akt, UMSC-PD-L1-HGF, ADSC-PD-L1-Akt, or BMSC-PD-L1-Akt into the carotid artery.

[0170] Firstly, it was demonstrated that intravenous or carotid artery injection of UMSC-PD-L1-Akt or UMSC-PD-L1-HGF (IV-UMSC-PD-L1-Akt, IV-UMSC-PD-L1-HGF, IA-UMSC-PD-L1-Akt, IA-UMSC-PD-L1-HGF) was more effective in reducing post-stroke infarct volume and maximum infarct area compared to IV-UMSC-Akt, IA-UMSC-Akt, IV-UMSC-HGF, IA-UMSC-HGF, IV-UMSC-PD-L1, IA-UMSC-PD-L1, IV-UMSCs, IA-UMSCs, and TTC-stained carrier control groups. Figure 3A-B). Then, rats treated with a combination of intracarotid and intravenous injections of UMSC-PD-L1-Akt or UMSC-PD-L1-HGF (IA-IV-UMSC-PD-L1-Akt or IA-IV-UMSC-PD-L1-HGF) showed mild infarction by TTC examination 3 days after cerebral ischemia. Figure 3C Quantitative measurements showed that, compared with the control group, the infarct volume was significantly reduced in rats treated with IA-IV-UMSC-PD-L1-Akt. Figure 3C Similarly, the maximum infarct area in rats treated with IA-IV-UMSC-PD-L1-Akt was smaller than that in rats treated with IA-IV-UMSC-Akt, IA-IV-UMSC-PD-L1, IA-IV-UMSCs, and the control group. Figure 3C Furthermore, to further determine the neuroregenerative effect 3 days after stroke, compared with IA-UMSC-PD-L1-Akt, IA-UMSC-PD-L1-HGF, IV-UMSC-PD-L1-Akt, IV-UMSC-PD-L1-HGF and the control group, the infarct volume and the largest infarct slice were significantly reduced in the IA-IV-UMSC-PD-L1-Akt or IA-IV-UMSC-PD-L1-HGF group. Figure 3D Similar results were also observed in rats treated with IA-IV-ADSC-PD-L1-Akt, IA-IV-BMSC-PD-L1-Akt, or IA-IV-BMSC-PD-L1-Akt. Figure 3E -F).

[0171] Example 5: Intracarotid artery combined with intravenous injection of UMSC-PD-L1-Akt, ADSC-PD-L1-Akt, or BMSC-PD-L1-Akt can improve the neurobehavioral response in stroke rats.

[0172] A stroke model demonstrated the in vivo neuroregenerative potential of IA-IV-UMSC-PD-L1-Akt. Two neurological deficit measures, including body asymmetry and motor activity, were assessed 28 days before and after stroke in rats. Rats were initially divided into four treatment groups: IA-IV-UMSC-PD-L1-Akt, IA-UMSC-PD-L1-Akt, IV-UMSC-PD-L1-Akt, and a control group. In the body asymmetry test, rats treated with IA-IV-UMSC-PD-L1-Akt showed better recovery than those treated with IA-UMSC-PD-L1-Akt, IV-UMSC-PD-L1-Akt, and the control group. Figure 4AFurthermore, compared with IA-UMSC-PD-L1-Akt, IV-UMSC-PD-L1-Akt, and the control group, IA-IV-UMSC-PD-L1-Akt rats also showed significant improvement in neurological deficits. Figure 4B These results indicate that IA-IV-UMSC-PD-L1-Akt has a higher neuroregenerative potential for stroke. Consistently, similar results were observed in rats treated with IA-IV-ADSC-PD-L1-Akt or IA-IV-BMSC-PD-L1-Akt. Figure 4C -D).

[0173] Example 6: UMSC-PD-L1-Akt treatment reduces neurological death after stroke

[0174] TUNEL staining was used to observe brain cell apoptosis in ischemic rats. In the control group (without stroke), almost no TUNEL staining was observed in any part of the brain. The penumbra region surrounding the ischemic core in the IA-IV-UMSC-PD-L1-Akt treatment group contained fewer TUNEL+ cells than those in the IA-IV-UMSC, IA-IV-UMSC-Akt, IA-IV-UMSC-PD-L1, and control groups. Figure 4E ).

[0175] Example 7: Targeting Study of UMSC-PD-L1-Akt-Luc in a Stroke Model

[0176] To demonstrate the homing effect of UMSC-PD-L1-Akt, the biodistribution of UMSC-PD-L1-Akt-Luc in the carotid artery or vein was studied using IVIS. UMSC-PD-L1-Akt-Luc transplanted intravenously was placed 6 hours post-injection and initially trapped in pulmonary capillaries, as shown in enhanced bioluminescence images of the lungs via IVIS ( ). Figure 5A In intracarotid artery injection within 24 hours post-stroke, UMSC-PD-L1-Akt-Luc homing did indeed survive and reposition to the right hemisphere of the stroke site within a period of 6 hours to over 1 week, without pulmonary uptake. Figure 5B In the IA-IV-UMSC-PD-L1-Akt-Luc group, the biodistribution of UMSC-PD-L1-Akt-Luc in the right hemisphere and lung was tracked from 6 hours to more than a week. Figure 5B Furthermore, in confocal microscopy immunofluorescence studies, luciferase+ cells located in the peri-infarct region showed co-localization with neuronal markers of MAP-2 or glial markers of GFAP. Figure 5C ).

[0177] Example 8: IA-IV-UMSC-PD-L1-Akt treatment can reduce the inflammatory response, but can increase CD8+ in ischemic brain. + CD122 + Tregs accumulation

[0178] To determine whether UMSC-PD-L1-Akt altered the composition of white blood cells after stroke, the absolute value of the total white blood cell count was measured. Figure 6A-1 and 6A-2 The gating strategy is shown. In the ischemic hemisphere of rats treated with UMSC-PD-L1-Akt, the total white blood cell count (including CD3+) was... + T cells, CD4 + T cells, CD11b + PD-L1 + Macrophages and F4 / 80 + PD-L1 + Microglia were significantly increased in both hemispheres, while the total number of cells in both hemispheres remained unchanged in the treatment and control groups. Figure 6B UMSC-PD-L1-Akt treatment in stroke rats significantly reduced activated CD11b in the ischemic hemisphere. + Tumor necrosis hormone-α (TNF-α), CD11b + INF-γ + CD3 + TNF-α + and CD3 + INF-γ + Percentage of cells ( Figure 6C This increases the total CD8+ in the ischemic hemisphere. + CD8 + CD122 + IL-10 + Treg cells and CD19 + IL-10 + Percentage of Breg cells ( Figure 6D-2 The production of interleukin-10 increased accordingly. Anti-PD-L1 monoclonal antibody treatment of CNS-infiltrating CD4+ cells in MCAO mice... + T cells were unaffected, but CD5 was observed. + CD1dhi-CD19 + Breg cells decreased.

[0179] Example 9: IA-IV-UMSC-PD-L1-Akt treatment increases the expression of regulatory molecules in spleen T cells after stroke.

[0180] To evaluate peripheral immune function after UMSC-PD-L1-Akt treatment, flow cytometry analysis was performed on the spleen of stroke-affected animals. Treatment with UMSC-PD-L1-Akt 4 hours post-stroke significantly suppressed CD19 levels in the spleen assessed 96 hours after stroke. + B cells, CD4 + and CD8 + T cells, CD11b + Monocytes / macrophages and CD11c + Expression of PD-L1 on dendritic cells (dc) Figure 7A-4 Conversely, UMSC-PD-L1-Akt significantly reduced activated CD3 in the spleen. + TNF-α + and CD3 + INF-γ + Percentage of inflammatory cells ( Figure 7B Furthermore, in stroke mice treated with UMSC-PD-L1-Akt, CD8+ was observed in the spleen. + CD122 + Tregs and CD19 + IL-10 + The percentage of Bregs increased ( Figure 7C Following UMSC-PD-L1-Akt treatment, Foxp3 + CD4 + CD25 + The frequency of Tregs remained unchanged.

[0181] Example 10: Administering UMSC-PD-L1-Akt, UMSC-PD-L1-HGF, ADSC-PD-L1-Akt, or BMSC-PD-L1-Akt via a combination of intravenous injections into the internal jugular vein alleviated left ventricular dysfunction and reduced the infarct size following myocardial infarction (MI).

[0182] To verify the important role of intravenous (IV) or intra-arterial (IA) UMSC-PD-L1-Akt or UMSC-PD-L1-HGF (IV-UMSC-PD-L1-Akt, IV-UMSC-PD-L1-HGF, IA-UMSC-PD-L1-Akt, IA-UMSC-PD-L1-HGF, IV-IA-UMSC-PD-L1-Akt or IV-IA-UMSC-PD-L1-HGF) in the rescue of myocardial ischemia injury, the infarct area in an AMI model was examined. At 28 days post-myocardial infarction, the infarct volume in either the IV-IA-UMSC-PD-L1-Akt treatment group or the IV-IA-UMSC-PD-L1-HGF treatment group was significantly smaller than that in the IV-UMSC-PD-L1-Akt, IV-UMSC-PD-L1-HGF, IA-UMSC-PD-L1-Akt, IA-UMSC-PD-L1-HGF and control groups. Figure 8A Furthermore, compared to the control group, the infarct wall thickness was increased in the IV-IA-UMSC-PD-L1-Akt treatment group or the IV-IA-UMSC-PD-L1-HGF treatment group. Figure 8B Similar results were also observed in rats treated with IV-IA-ADSC-PD-L1-Akt or IV-IA-BMSC-PD-L1-Akt. Figure 8C -D).

[0183] Example 11: Anti-inflammatory effect of UMSC-PD-L1-Akt on ischemic myocardium

[0184] To observe whether UMSC-Akt-PD-L1 treatment suppressed the inflammatory response after myocardial infarction (MI), immunohistochemical analysis was performed on inflammatory cell infiltration 3 days after MI. Compared with IV-UMSC-PD-L1-Akt, IA-UMSC-PD-L1-Akt, and the control group, the inflammation in the IV-IA-UMSC-PD-L1-Akt treatment group was significantly reduced (…). Figure 9A Compared with the IV-UMSC-PD-L1-Akt group and the control group, the IV-IA-UMSC-PD-L1-Akt, IA-UMSC-PD-L1-Akt treatment groups and the control group showed significantly reduced CD68+ cell infiltration in the periinfarct area 3 days after infarction. Figure 9B ).

[0185] Example 12: UMSC-PD-L1-Akt treatment can alleviate MI-induced fibrosis

[0186] Compared with sham surgery, 28 days after myocardial infarction, trichrome stained sections showed a significant increase in left ventricular fibrosis. Figure 9CInterestingly, compared with IV-UMSC-PD-L1-Akt, IA-UMSC-PD-L1-Akt, and the control group, we observed a significant reduction in post-myocardial infarction fibrosis after IV-IA-UMSC-PD-L1-Akt treatment. Figure 9C ).

[0187] Example 13: Targeting Study of UMSC-PD-L1-Akt-Luc in an Acute Myocardial Infarction Model

[0188] To demonstrate the homing effect of UMSC-PD-L1-Akt, the biodistribution of UMSC-PD-L1-Akt-Luc in the carotid artery or vein was studied using IVIS. Intravenously injected UMSC-PD-L1-Akt-Luc transplantation began to initially trap in pulmonary capillaries 6 hours after injection, as shown in enhanced bioluminescence images of the lungs via IVIS ( Figure 10 In intracarotid artery injection 24 hours after myocardial infarction, UMSC-Akt-PD-L1-Luc homing did indeed survive and migrate to the cardiac region without pulmonary uptake over a period of 6 hours to more than 1 week. Figure 10 ).

[0189] While this disclosure has been described in conjunction with the specific embodiments described above, many alternatives, modifications, and variations thereof will be apparent to those skilled in the art. All such alternatives, modifications, and variations are considered to fall within the scope of this disclosure.

Claims

1. A genetically engineered mesenchymal stem cell population (MSCs), wherein the MSCs contain a survival gene and an immune checkpoint gene, wherein the MSCs are genetically engineered by the survival gene of Akt or hepatocyte growth factor (HGF) and the immune checkpoint gene of PD-L1, wherein the genetic engineering modification is gene transfection.

2. The genetically engineered MSCs as described in claim 1, wherein the MSCs are umbilical cord mesenchymal stem cells (UMSCs), adipose-derived mesenchymal stem cells (ADSCs), or bone marrow mesenchymal stem cells (BMSCs).

3. The genetically engineered MSCs as described in any one of claims 1 or 2, wherein the survival gene and the immune checkpoint gene are contained in a vector.

4. The genetically engineered MSCs as described in claim 3, wherein the vector is a lentiviral vector.

5. A pharmaceutical composition comprising MSCs as described in any one of claims 1 to 4.

6. A method for synergistically increasing the survival status and immunomodulatory capacity of MSCs or enhancing the proliferation of MSCs in vitro, comprising transfecting MSCs with synergistically effective amounts of the survival gene of Akt or hepatocyte growth factor HGF and the immune checkpoint gene of PD-L1.

7. The method of claim 6, wherein the MSCs are UMSCs, ADSCs, or BMSCs.

8. Use of the genetically engineered MSCs as described in any one of claims 1 to 4, for the manufacture of agents for the prevention, improvement and / or treatment of stroke.

9. The use of genetically engineered MSCs as described in any one of claims 1 to 4, wherein the MSCs are used to manufacture agents for the prevention, improvement and / or treatment of myocardial infarction (MI).

10. The use as described in claim 8 or 9, wherein the MSCs are UMSCs, ADSCs, or BMSCs.

11. The use as described in claim 8 or 9, wherein the effective amount of the agent is in the range of 1 × 10⁻⁶. 5 One cell to 1×10 8 Each cell.

12. The use as described in claim 8 or 9, wherein the agent reduces the inflammatory response but enhances CD8+ in ischemic tissue. + CD122 + Accumulation of Tregs.

13. The use as described in claim 8 or 9, wherein the agent increases the expression of regulatory molecules on T cells in an individual.

14. The use as described in claim 9, wherein the MI is an acute myocardial infarction (AMI).

15. The use as described in claim 8 or 9, wherein the agent is suitable for intravenous injection, intra-arterial injection, or a combination thereof.

16. The use as described in claim 15, wherein the intra-arterial injection is an intra-carotid artery injection.

17. The use as described in claim 8 or 9, wherein the agent is suitable for intracarotid artery injection combined with intravenous injection.

18. The use as described in claim 8 or 9, wherein the agent is suitable for intra-arterial injection combined with intravenous injection.

19. The use as described in claim 8 or 9, wherein in an individual suffering from stroke or AMI, the agent is suitable for intracarotid artery injection combined with intravenous injection.

20. The use as described in claim 8 or 9, wherein in an individual suffering from stroke or AMI, the agent is suitable for intra-arterial injection combined with intravenous injection.

21. The use as described in claim 19, wherein the effective amount of the agent is 1 × 10⁻⁶ mmol / L injected into the carotid artery. 4 Cells up to 1×10 6 Cells, intravenous injection 3×10 4 Cells up to 1×10 7 cell.

22. The use as described in claim 8 or 9, wherein the agent reduces MI-induced fibrosis, reduces inflammation in ischemic tissue, reduces functional impairment after MI and reduces infarct size after MI, increases the expression of regulatory molecules on T cells in the spleen after stroke, or reduces neuronal death in stroke-induced brain injury.

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