Platelet-derived mitochondrial therapy and methods for generating pluripotent cells

Abstract

This method generates pluripotent stem cells from adult human peripheral blood cells by isolating peripheral blood insulin-producing cells and exposing them to adult peripheral blood-derived mitochondria. Adult peripheral blood insulin-producing cells (PB-IPCs) are isolated from adult peripheral blood by attachment to a positively charged hydrophobic surface, such as a Petri dish. Once isolated, adult peripheral blood-derived mitochondria are applied to the isolated PB-IPCs. The mitochondria are then taken up by the PB-IPCs and enter their nuclei, reprogramming the cells and converting them into pluripotent stem cells, generating cells derived from three germ layers. Furthermore, PB-IPCs give rise to functional CD34+ hematopoietic stem cell (HSC)-like cells after treatment with adult peripheral blood-derived mitochondria.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 976,830, filed February 14, 2020, which is incorporated herein by reference in its entirety.

[0002] Reference to the sequence listing provided by EFS-WEB The Contents Sequence Listing "5700NP_and_PCT_Sequence_Listing_ST25" ASCII text file is 1,595 KB in size, was created on February 15, 2021, and was filed electronically with the specification via EFS-Web, and is incorporated herein by reference in its entirety.

[0003] The present invention relates generally to medical therapy, and more specifically to treating human adult peripheral blood cells with platelet-derived mitochondria to generate pluripotent cells for the treatment of chronic medical conditions. [Background technology]

[0004] Many chronic medical conditions, including but not limited to cancer, Alzheimer's disease, and diabetes, affect the health of millions of people every day, yet available treatments for many of these conditions remain largely ineffective. Diabetes is a major public health concern, affecting more than 350 million people worldwide. The prevalence of diabetes is 12.1% of the population in India, 11.6% in China, and over 9.3% in the United States, with approximately 1 billion people worldwide considered prediabetic, meaning they have higher than normal blood sugar levels. Diabetes is the sixth leading cause of death in the United States and is associated with an increased risk of heart disease, stroke, kidney disease, blindness, and amputation.

[0005] A common factor in both type 1 diabetes (T1D) and type 2 diabetes (T2D) is immune system dysfunction. T1D is characterized by autoimmune destruction of pancreatic islet β cells and destruction of immune cells, including T cells, B cells, regulatory T cells (Tregs), monocytes / macrophages (Mo / Mφ), dendritic cells (DCs), natural killer (NK) cells, and natural killer T (NKT) cells. T2D is primarily characterized by insulin resistance and abnormal insulin production, but low-grade chronic inflammation also occurs in peripheral tissues such as adipose tissue, liver, and muscle tissue, further contributing to the disease. Specifically, studies have shown that T cells are unexpected promoters and regulators of insulin resistance. T cells promote the recruitment of inflammatory macrophages to fat depots, resulting in the production of inflammatory cytokines, which promote the development of insulin resistance, which can lead to diabetes. Despite more than 30 years of intense research, treatments for both type 1 and type 2 diabetes remain elusive. To simultaneously address the multiple immune dysfunctions underlying these diseases, comprehensive immunomodulation is required through both local and systemic approaches.

[0006] Deficiency of insulin-producing cells is another serious and common problem for diabetic patients. While insulin replacement provides T1D patients with a means of managing blood glucose, it is not a cure, and insulin does not address the underlying immune dysfunction that causes pancreatic islet β-cell destruction. To overcome the deficiency of insulin-producing cells in diabetic patients, pancreas and islet transplantation offers the potential for treatment independence from insulin injections. However, donor shortages and the risk of immune rejection significantly hinder the potential for widespread application of such transplants. Therefore, there remains a compelling need and urgency to find a diabetes treatment that not only halts the progression of autoimmunity in T1D and corrects multiple immune dysfunctions in T2D, but also overcomes the deficiency of insulin-producing β-cells.

[0007] Stem cell research has the potential to revolutionize the treatment of certain life-altering injuries and human diseases, including, but not limited to, diabetes, Alzheimer's disease, cancer, and alopecia areata. To date, researchers have characterized multiple types of human stem cells with various regenerative capabilities, and animal studies and human clinical trials have demonstrated the translational potential of stem cells to treat human diseases.

[0008] To date, functional insulin-producing cells or pancreatic islet cells have been generated from embryonic stem (ES) cells and induced pluripotent stem (iPS) cells by ex vivo differentiation. However, recent advances in stem cell biology have recognized that ES cells, iPS cells, and their derived cells may also cause immune rejection after transplantation, challenging their clinical therapeutic potential. Therefore, in an effort to avoid immune cell attack on transplanted cells and / or cell delivery devices and to provide sufficient permeabilized nutrients to maintain cell viability, encapsulation with different biomaterials and the use of semipermeable membranes and capsules have been evaluated in animal and pilot clinical trials. However, fibrosis formation, or scarring around the device that causes encapsulated cell death and device failure, remains a major obstacle, and an optimal membrane or capsule device has yet to be developed.

[0009] For over 30 years, the most common stem cell therapy approved by the U.S. Food and Drug Administration (FDA) has been hematopoietic cell transplantation (HCT) (also known as hematopoietic stem cell transplantation or HSCT). HCT has been approved for the treatment of bone marrow failure, malignant blood disorders, genetically-based blood disorders, and autoimmune diseases, as well as for cellular regeneration after chemotherapy and / or radiation. However, several major limitations have limited the widespread clinical application of allogeneic HCT. These limitations include the difficulty of identifying fully human leukocyte antigen (HLA)-matched or haploidentical donors; the scarcity of hematopoietic stem cells (HSCs), which are known to be identified by the glycosylated transmembrane protein marker CD34 across all sources of collected cells (≤1%), particularly due to the occurrence of graft-versus-host disease (GVHD), opportunistic infections, relapse of the primary disease, and toxicities associated with immunosuppressants and radiation. While an autologous source of HSCs would address the issues of matching and GVHD, engraftment remains limited by the CD34 marker. + It may still be hampered by the limited number of HSCs.

[0010] Clinical HCT engraftment success rates are significantly higher in patients with functional CD34 + To correlate the numbers of hematopoietic progenitor cells (HPCs) and HSCs, researchers have evaluated whether embryonic stem (ES) cells and / or induced pluripotent stem (iPS) cells can be manipulated to generate HSCs by reprogramming with small molecules or viral transduction of transcription factors. To date, these approaches have been limited by an inability to generate truly functional HSCs in sufficient numbers for therapeutic use, safety and ethical concerns, and potential immune rejection of ES or iPS derivatives.

[0011] The use of autologous stem cells for regenerative medicine would be more ethically acceptable and likely to be successful than the use of other stem cells, and such therapies would avoid many of the immune rejection and safety concerns associated with other stem cell (e.g., ES- or iPS-based) therapies. What is needed is a method for generating autologous pluripotent cells for use in regenerative medicine.

[0012] Heretofore, no method has been available for generating pluripotent cells for medical treatment that has the advantages and features of the present invention. Summary of the Invention

[0013] The present invention discloses a method for generating pluripotent stem cells from adult human peripheral blood cells by isolating peripheral blood insulin-producing cells and exposing them to platelet-derived mitochondria. In one embodiment of the present invention, adult peripheral blood insulin-producing cells (PB-IPCs) are isolated from adult peripheral blood via attachment to a positively charged hydrophobic surface. Once isolated, platelet-derived mitochondria are applied to the isolated PB-IPCs. The mitochondria are then incorporated into the PB-IPCs and enter their nuclei, thereby reprogramming the cells and converting them into pluripotent stem cells, giving rise to cells derived from three germ layers.

[0014] PB-IPCs can be easily isolated from peripheral blood and expanded in serum-free medium, avoiding the painful and invasive procedure required for bone marrow harvest. Mitochondrial therapy using autologous PB-IPCs from patients as a starting material can generate functional autologous mitochondria-induced peripheral blood insulin-producing cells (miPB-IPCs) on a large scale and give rise to different cell lineages. Highly efficient differentiation of these miPB-IPCs into multiple lineages, including T cells, B cells, monocytes / macrophages (Mφ), granulocytes (Gr), erythrocytes (Er), megakaryocytes (MK) / platelets, retinal pigment epithelium (RPE), and neurons, demonstrates the multipotency of PB-IPC mitochondrial reprogramming. These cells therefore hold great promise as a solution to the current bottleneck associated with conventional stem cell transplantation and have great potential for patient benefit in the clinic.

[0015] PB-IPCs naturally circulate in human peripheral blood, express islet β cell-associated markers, and migrate to pancreatic islets to alleviate hyperglycemia. After optimizing ex vivo culture conditions, the present invention provides a novel approach for generating large quantities of autologous insulin-producing cells from a patient's own blood, potentially treating diabetes in the clinic. In contrast to generating insulin-producing cells from ES cells and iPS cells, the present technology can efficiently isolate insulin-producing cells from a patient's own blood without any ethical issues or risk of immune rejection. Furthermore, pluripotent differentiation of miPB-IPCs into other cell lineages addresses the treatment of not only diabetic patients, but also the entire field of regenerative medicine.

[0016] In another embodiment of the present invention, PB-IPCs give rise to functional CD34+ hematopoietic stem cell (HSC)-like cells after treatment with platelet-derived mitochondria, herein referred to as mitochondria-induced CD34+ HSCs (miCD34+ HSCs). The miCD34+ HSCs of the present invention can reconstitute multilineage blood cells, including, but not limited to, T cells (CD3+CD4+), B cells (CD19+), monocytes / macrophages (CD14+), granulocytes (CD66b+), erythroid cells (CD235a+), and megakaryocytes / platelets (CD41b+), in peripheral blood, spleen, and bone marrow 12 weeks after transplantation into irradiated NSG mice. This highlights their potential for treating hematopoietic-related diseases.

[0017] Self-renewal is one of the key properties of CD34+ HSCs. miCD34+ HSCs exhibit limited self-renewal but rapid pluripotency, which implies differentiation of PB-IPCs into CD34+ HSC-like cells through mitochondrial reprogramming, expression of HSC-associated cell surface markers, and primarily improved pluripotency, without significantly altering their self-renewal capacity.

[0018] The drawings constitute a part of this specification and include illustrative embodiments of the present invention, illustrating various objects and features of the present invention. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 is a schematic diagram showing a method for generating pluripotent cell differentiation by treating peripheral blood insulin-producing cells with platelet-derived mitochondria, which method embodies the present invention. [Figure 2] FIG. 1 is a schematic diagram showing a method and animal protocol embodying one embodiment of the present invention for in vivo multipotency of mitochondrially-derived CD34+ hematopoietic stem cell-like cells after transplantation into irradiated NSG mice. [Figure 3]Flow cytometry graphs showing the characteristics of peripheral blood-derived insulin-producing cells (PB-IPCs) from adult peripheral blood with islet β cell-associated markers, demonstrating that gated Lin1-CD34- cells express CD45, SOX2, CD45RO, and CCR7 (n=8). [Figure 4] Flow cytometry graphs showing the phenotype of PB-IPCs are shown, showing low expression of CD117 and absence of CD4, CD8, CD19, CD34, CD38, CD41, CD42a, and CD66b. Isotype-matched IgG was used as a control (n=8). [Figure 5] Figure 1 shows apoptosis (Annexin V+) and necrosis (7-AAD+) of blood monocytes after 24 hours of culture in untreated tissue culture Petri dishes (n=5). [Figure 6] FIG. 1 shows analysis of the cell cycle in freshly isolated PB-IPCs after overnight attachment by flow cytometry and propidium iodide (PI) staining (n=5). [Figure 7] Figure 1 shows real-time PCR analysis of islet β cell-associated markers in PB-IPCs isolated from healthy donors (n=5). Freshly isolated human islets were used as a positive control. [Figure 8] FIG. 1 shows flow cytometry results of double immunostaining for the pancreatic islet β cell-associated transcription factor MAFA and the insulin by-product C-peptide (n=5). [Figure 9] Flow cytometry to determine the phenotype of PB-IPCs after overnight adherence. Representative data shown from four preparations. [Figure 10] Fluorescence microscopy images showing GFP+ cells in PBMCs of insulin promoter 1-GFP transgenic mice (n=3). GFP-positive mouse islets were used as a positive control. [Figure 11] Phase contrast images showing differentiation of miPB-IPCs into RPE cells, with cellular pigmentation and processes of various lengths (n=5). [Figure 12]Immunostaining of differentiated RPE cells with RPE-specific markers (n=3). Human primary RPE cells were used as a positive control. Mouse IgG and rabbit IgG were combined with nuclear DAPI (blue) staining and used as negative controls (inset). Untreated miPB-IPCs were used as a negative control (center panel). [Figure 13] Figure 1 shows the phagocytosis of fluorescent beads by differentiated RPE cells (n=3). [Figure 14] Flow cytometry analysis of CD36 expression in differentiated RPE cells and untreated cells (n=3). Isotype-matched IgG was used as a negative control. [Figure 15] Phase contrast images showing differentiation of miPB-IPCs into neurons (n=5). [Figure 16] Immunostaining of differentiated neuronal cells with the neuron-specific markers synapsin I and tyrosine hydroxylase (n=3). IgG staining was used as a negative control (inset). Untreated miPB-IPCs were used as a negative control (top panel). [Figure 17] FIG. 1 shows colony formation of miPB-IPCs with different sizes in normal miPB-IPC cell culture (n=5). [Figure 18] Graph showing differences in colony formation potential between miPB-IPC compared to untreated PB-IPC. Data are presented as mean±SD from five preparations. [Figure 19] FIG. 1 shows phenotypic analysis of single colony-derived cells harboring PB-IPC markers CD34-CD45+SOX2+CD45RO+CCR7+ (n=5). [Figure 20] Clonal analysis: Single colonies were dispersed and seeded into 96-well plates, and wells were treated with different lineage-specific inducers for differentiation, including macrophages (left, phagocytosis of fluorescent beads, n=3), RPE cells (center, n=6), and neurons (right, n=9). [Figure 21]Figure 1 shows weight gain in miPB-IPC-implanted mice without tumor formation. NOD-scid IL-2Rγ null mice (n=3) were inoculated with miPB-IPC at a dose of 2x10 cells / mouse (sc, right lower flank). An injection of the same volume of saline into the left lower flank served as a control. [Figure 22] Colony analysis images using three germ layer-related markers (ectodermal neuron marker synapsin I, endodermal pancreatic islet β cell marker insulin, and mesodermal macrophage marker CD11b). IgG was used as a negative control (top panel). Representative images were from one of eight colonies in the miPB-IPC group (bottom panel) and one of five colonies in the control PB-IPC group (middle panel). [Figure 23] Colony analysis images using three additional germ layer-associated markers: the neuronal marker βIII tubulin (Tuj1) for ectoderm, the hepatocyte marker alpha-fetoprotein (AFP) for endoderm, and smooth muscle actin (SMA) for mesoderm. IgG was used as a negative control (top panel). Representative images were from one of seven colonies in the miPB-IPC group (bottom panel) and one of five colonies in the control PB-IPC group (middle panel). [Figure 24] 10 is a transmission electron microscope image showing mitochondria (M) at the nuclear membrane of mitochondria-treated PB-IPC. [Figure 25] Transmission electron microscope image showing mitochondria located inside the nuclear matrix and close to the nucleolus with morphologically similar mitochondria (indicated by arrows) in the cytoplasm. [Figure 26] FIG. 1 shows the ultrastructure of untreated PB-IPCs. [Figure 27]Figure 1 shows penetration of red fluorescent protein (RFP)-labeled mitochondria into PB-IPCs. After PB-IPCs were treated with RFP-labeled mitochondria for 4 hours, RFP+ mitochondria infiltrating into the cytoplasm were confirmed by confocal microscopy (n=5). The distribution of RFP+ mitochondria within the nucleus is indicated by arrows. Hoechst33342-labeled nuclei and differential interference contrast (DIC) images (left) demonstrate the colocalization of RFP+ mitochondria. [Figure 28] Figure 1 shows MitoTracker Red-labeled mitochondria that have entered the nucleus, and their colocalization is demonstrated by confocal microscopy (n=5). Mitochondria isolated from platelets were co-cultured with purified nuclei from PB-IPCs in the presence of serum-free medium X-VIVO15 at 37°C and 5% CO for 4 hours. [Figure 29] FIG. 1 shows the expression of CXCR4 on the membrane of purified nuclei (n=4). [Figure 30] FIG. 1 shows mitochondria presenting the CXCR4 ligand SDF-1 (n=4). [Figure 31] Figure 1 shows a blocking experiment with the CXCR4 receptor antagonist AMD3100. Purified PB-IPC nuclei were treated with MitoTracker Red-labeled purified mitochondria with or without AMD3100 (30 μM, n=3). An equivalent concentration of the solvent DMSO was used as a control. After 4 h of treatment, nuclei were washed twice with PBS and prepared for flow cytometry. [Figure 32] (B) Flow cytometry showing SDF-1 expression in platelet-, PBMC-, and PB-IPC-derived mitochondria. Isotype-matched IgG was used as a control. Data are representative of three experiments. [Figure 33] A graphical comparison of SDF-1 expression levels between platelet-derived mitochondria (Plt-Mito) and PBMC-derived mitochondria (PBMC-Mito) is shown, with no significant difference, but SDF-1 expression in PB-IPC-derived mitochondria (PB-IPC-Mito) is much lower. Data represent mean ± SD. n=3. [Figure 34] 10A-10C are confocal microscopy images showing penetration of PBMC-derived mitochondria into the nuclei of PB-IPCs. [Figure 35] Figure 1 shows a real-time PCR array of epigenetic chromatin modification enzyme-related genes. [Figure 36] FIG. 1 shows an RNA-seq heatmap depicting 46 differentially expressed genes in PB-IPCs after mitochondrial treatment. [Figure 37] FIG. 1 shows RNA-seq data showing 37 upregulated genes in PB-IPCs after mitochondrial treatment. [Figure 38] FIG. 1 shows RNA-seq data showing nine down-regulated genes in PB-IPCs after mitochondrial treatment. [Figure 39] Figure 1 shows a histogram of the purity of isolated mitochondria. Different markers, including MitoTrack Deep Red staining, anti-cytochrome C, and anti-heat shock protein (HSP), were applied by flow cytometry. 60 antibodies were used for mitochondrial markers, calnexin for the endoplasmic reticulum (ER), and GM130 for the Golgi apparatus. Isotype-matched IgG was used as a negative control (n=3). [Figure 40] Figure 1 shows upregulation of CD34 expression after mitochondrial treatment in miPB-IPCs. Data represent the mean ± SD of five experiments. [Figure 41] Phenotypic characterization of gated miCD34+ HSCs (dashed arrows) with additional surface markers in total miPB-IPCs. Isotype-matched IgG was used as a control. Data are representative from five preparations. [Figure 42] Phenotypic characterization of gated CD34+CD45RA- HSCs (bottom) and CD34+CD45RA+ cell populations with additional markers (top) in total PBMCs (n=4). Isotype-matched IgG was used as a control. Data are representative from one of four preparations. [Figure 43] 1 shows a schematic flow chart outlining the protocol of the present invention from generation of miPB-IPCs to pluripotent cell differentiation of miPB-IPCs. miCD34+ HSCs were purified for in vitro and in vivo differentiation, respectively. [Figure 44] Phase-contrast images showing T cell differentiation of purified miCD34+ HSCs (n=4) in the presence of FLT-3 ligand, IL-2, and IL-7 for 3 days. Untreated cells were used as a control (left). Treated CD34+ HSCs showed significant morphological changes, with cell clusters (stars) and some floating cells (arrows) released from the cell clusters (stars). Magnification: ×200. [Figure 45] Phase contrast images showing the morphology of control PB-IPCs (left) and PB-IPCs treated in the presence of mitochondria (right) (n=4). Original magnification = ×200. [Figure 46] Figure 1 shows z-stack confocal images (n=4) showing strong expression of human T cell markers CD4 and low expression of CD8. Untreated miCD34+ HSCs were used as a control for immunostaining (left panel). [Figure 47] Flow cytometry shows that differentiated T cells are CD3+CD4+CD8-CD38+ (n=4). Untreated miCD34+ HSCs were used as a control (top panel). [Figure 48] FIG. 1 shows the expression of T cell receptor α / β (TCRαβ) in gated CD3+ and CD4+ T cells (n=4). [Figure 49] Figure 1 shows intracellular staining of differentiated T cells using Th1 / Th2 cell cytokine markers (n=3). Isotype-matched IgG was used as a control. Data are presented as the mean ± SD of three experiments. [Figure 50] Figure 1 shows the fold change in cytokine expression levels in in vitro differentiated T cells after stimulation with PMA and ionomycin (n=3). Data represent the mean ± SD. [Figure 51](Figure 1 shows the differentiation of miCD34+ HSCs into macrophages after 3 days of treatment with 50 ng / mL M-CSF. Differentiated Mφs displayed macrophage markers CD11b and CD209 and exhibited phagocytosis of fluorescent beads (n=4). Untreated miCD34+ HSCs served as a control. [Figure 52] miCD34+ HSCs were differentiated into granulocytes after treatment with 100 ng / mL G-CSF and 25 ng / mL FLT-3L for 3 days, followed by Wright-Giemsa staining for the granulocyte marker CD66b (left) and flow cytometry (n=4). Untreated miCD34+ HSCs served as a control. [Figure 53] FIG. 1 shows the differentiation of miCD34+ HSCs into erythroid cells as shown by phase contrast imaging of mature RBCs (indicated by arrows) and Wright-Giemsa staining of the typical morphology of mature RBCs (indicated by arrows) (n=4). [Figure 54] FIG. 1 shows analysis of the percentage of mature CD45 − hemoglobin + RBCs among gated CD235a + cells by flow cytometry (n=4). [Figure 55] FIG. 1 shows differentiation of miCD34+ HSCs into megakaryocytes / platelets after 7 days of treatment with FLT-3L+TPO, showing CD42+ and multinucleated appearance (n=4). [Figure 56] Polyploid MK analysis after 7 days of treatment with FLT-3L + TPO is shown by histogram (center) and dot plot (top) of MK / platelet marker CD42a. Normal platelets and T cells from healthy donors were used as controls for cells without nuclei (0N) and cells with one nucleus (1N) (bottom) (n=4). [Figure 57] FIG. 1 shows Wright-Giemsa staining showing differentiated miCD34+ HSCs with multiple nuclei (indicated by arrows) after treatment with FLT-3L+TPO for 7 days (n=4). [Figure 58]Figure 1 shows the engraftment levels of hCD45+mCD45.1- cells in the peripheral blood, spleen, and bone marrow of miCD34+ HSC-transplanted NSG mice at 12 weeks (3 x 105 cells / mouse in 200 µL saline, i.e., n = 6). [Figure 59] Phenotypic characterization of miCD34+ HSCs after engraftment into irradiated NSG mice (3x105 cells / mouse in 200µL saline, iv, n=5). Tissue samples were collected 12 weeks post-transplant. After excluding propidium iodide (PI)-positive dead cells, gating was performed to analyze only live cells (histograms). Gated live cells positive for human leukocyte common antigen CD45 and negative for mouse CD45.1 were analyzed. Representative data are from one of three experiments with similar results. Isotype-matched IgG was used as a control for flow cytometry. [Figure 60] FIG. 1 shows multilineage differentiation of miCD34+ HSCs 12 weeks after transplantation into irradiated NSG mice. [Figure 61] Erythroid reconstitution of miCD34+-transplanted NSG mice at 12 weeks (n=6). After excluding propidium iodide (PI)-positive dead cells, gating was performed to analyze only live cells (histograms). SYTO60 was used to stain CD235a+ nucleated erythroid cells. [Figure 62] Characterization of miCD34+ HSCs after 12 weeks of engraftment in irradiated NSG mice (n=5). Representative data are from one of three experiments with similar results. Gated human CD45-positive and mouse CD45.1-negative live cells were analyzed. Isotype-matched IgG was used as a flow cytometry control. [Figure 63] Figure 1 shows myeloid differentiation of miCD34+ HSCs after transplantation into NSG mice at 12 weeks (top) and 16 weeks (bottom, n=3). [Figure 64] FIG. 1 shows analysis of Notch ligands on platelet-derived mitochondria by flow cytometry (n=3). [Figure 65]Figure 1 shows the expression of Notch receptors on PB-IPCs by flow cytometry. PB-IPCs were treated with mitochondria for 7 days and collected for flow cytometry. Untreated PB-IPCs were used as a control. n=3. [Figure 66] Phase contrast images showing the morphology of PB-IPCs (left) and PB-IPCs treated in the presence of mitochondria (center) and mitochondria plus DAPT (right). Original magnification = x200. [Figure 67] Figure 1 shows upregulation of CD34 expression after treatment with mitochondria and / or DAPT. DAPT-treated PB-IPCs were used as controls. n=4. Data represent mean ± SD. DETAILED DESCRIPTION OF THE INVENTION

[0020] I. Introduction and Environment Where necessary, detailed embodiments of the present invention are disclosed herein, but it will be understood that the disclosed embodiments are merely examples of the invention, which may be embodied in various and alternative forms. Accordingly, the specific structural and functional details disclosed herein should not be construed as limiting, but should be used merely as a basis for the claims and as a representative basis for teaching those skilled in the art how to use the present invention in a variety of ways, in virtually any appropriately detailed configuration.

[0021] In the following description, for convenience, certain terminology is used by way of reference only and not by way of limitation. For example, top, bottom, front, rear, right, and left refer to the invention as oriented toward the view being referenced. The words "inwardly" and "outwardly" refer to directions toward and away from, respectively, the geometric center of the embodiment being described and designated portions thereof. Where appropriate, forward and backward generally refer to the direction of travel. This term includes the specifically mentioned word, derivatives thereof, and words of similar import.

[0022] II. Preferred Embodiments The present invention discloses a method for generating pluripotent stem cells from adult human peripheral blood cells by isolating peripheral blood insulin-producing cells (PB-IPCs) and exposing them to platelet-derived mitochondria. PB-IPCs, also known as "peripheral blood stem cells (PB-SCs)," are further characterized by Zhao et al., U.S. Patent No. 8,835,163, which is incorporated herein by reference in its entirety. Figure 1 shows a schematic diagram of an embodiment of the inventive protocol.

[0023] PB-IPCs have been demonstrated to exist in human peripheral blood and exhibit a unique phenotype (Lin1 - CD34 - CD45 + CD45RO + CCR7 + SOX2 + OCT3 / 4 + MAFA + Glut2 + ) is shown. In an exemplary embodiment, a sample of adult human peripheral blood is first obtained and centrifuged. After centrifugation, peripheral blood-derived mononuclear cells (PBMCs) are isolated from the adult peripheral blood. PB-IPCs are then isolated from the PBMCs via adherence to a positively charged hydrophobic surface. In a preferred embodiment, PB-IPCs are isolated, grown, and expanded by adhering to the hydrophobic bottom of a Petri dish in a chemically defined, serum-free culture without the addition of any other growth factors. However, in alternative embodiments, other devices and / or media with hydrophobic surfaces can be utilized to isolate PB-IPCs.

[0024] In an exemplary embodiment, a sample of adult human peripheral blood platelets is obtained. In an embodiment of the present invention, the platelet sample can be collected from the same peripheral blood sample as the isolated PB-IPCs or from a different adult human peripheral blood source. Mitochondria are then isolated from the peripheral blood platelets. Platelet-derived mitochondria can be isolated as described herein or by alternative mitochondrial isolation methods.

[0025] After PB-IPCs and platelet-derived mitochondria are isolated, the platelet-derived mitochondria are applied to the isolated PB-IPCs. During this procedure, the mitochondria are taken up by the PB-IPCs, thereby reprogramming the cells and converting the PB-IPCs into pluripotent stem cells, resulting in cells derived from three germ layers. Upon invading the PB-IPCs, a portion of the applied platelet-derived mitochondria enters the nuclei of the PB-IPCs, corresponding to the cellular reprogramming.

[0026] The differentiation potential of PB-IPCs significantly increased after treatment with platelet-derived mitochondria, resulting in cells derived from three germ layers. Mitochondrial-induced PB-IPCs (miPB-IPCs) showed high differentiation efficiency into RPE and neural cells in the presence of different inducers, confirming the multipotency of PB-IPCs after mitochondria treatment. Therefore, these cells hold great promise as a solution to the current bottleneck associated with conventional stem cell transplantation and have great potential for patient benefit in the clinic.

[0027] PB-IPCs naturally circulate in human peripheral blood, express islet β cell-associated markers, and migrate to pancreatic islets to alleviate hyperglycemia after transplantation into diabetic mice induced with the chemical streptozotocin (STZ). Therefore, the present invention provides a novel approach for generating large quantities of autologous insulin-producing cells from a patient's own blood, potentially treating autoimmune diseases in the clinic. Compared to generating insulin-producing cells from ES cells and iPS cells, the present technology can efficiently isolate insulin-producing cells from an individual's own blood without any ethical issues or risk of immune rejection. Furthermore, the pluripotent differentiation of miPB-IPCs into other cell lineages circumvents these limitations, creating a previously unmet medical need not only for autoimmune disease patients but for the entire field of regenerative medicine. In embodiments of the present invention, miPB-IPCs can be differentiated into macrophage cells, neuronal cells, RPE cells, granulocyte cells, T cells, B cells, erythrocytes, megakaryocytic cells, platelet cells, bone marrow cells, stromal cells, osteoblasts, keratinocytes, hair follicle cells, glandular cells, endothelial cells, corneal endothelial cells, cardiac myocytes, muscle cells, epithelial cells, hepatocytes, kidney cells, pancreatic islet beta cells or other cell types when exposed to the appropriate promoters, respectively.

[0028] In an alternative embodiment, mitochondria derived from PBMCs (not platelets) are isolated and applied to PB-IPCs. During treatment, the mitochondria from PBMCs invade PB-IPCs and penetrate into the nuclei of PB-IPCs, corresponding to cellular reprogramming. In a further embodiment of the present invention, mitochondria can be isolated from plasma, serum, or other parts of human blood and used to treat PB-IPCs.

[0029] In another aspect of the invention, PB-IPCs give rise to functional CD34+ hematopoietic stem cell (HSC)-like cells after treatment with platelet-derived mitochondria, referred to herein as mitochondrial-derived CD34+ HSCs (miCD34+HSCs). Mitochondrial therapy uses autologous PB-IPCs from patients as starting material, and functional autologous mitochondrial-derived CD34+ HSCs are generated. + (miCD34 +) Hematopoietic stem cells (HSCs) are generated on a large scale, giving rise to various blood lineages. The isolation and expansion of PB-IPCs from peripheral blood is performed using serum-free culture media with a hydrophobic surface, avoiding the painful and invasive procedure required for bone marrow removal. Isolated platelet-derived mitochondria are applied to PB-IPCs, enter the PB-IPCs, and then enter the nuclei of the PB-IPCs, allowing for cellular reprogramming. The mitochondria-induced PB-IPCs are then further exposed to a hematopoietic promoter for HSC-like differentiation.

[0030] The animal protocol for carrying out the present invention is shown in Figure 2. After transplantation into irradiated NSG mice, the miCD34+ HSCs of the present invention can reconstitute multilineage blood cells, including, but not limited to, T cells (CD3+CD4+), B cells (CD19+), monocytes / macrophages (CD14+), granulocytes (CD66b+), erythroid cells (CD235a+), and megakaryocytes / platelets (CD41b+), in the peripheral blood, spleen, and bone marrow at 12 weeks. This indicates the high potential of the present invention for treating hematopoietic-related diseases.

[0031] Self-renewal capacity is one of the key characteristics of CD34+ HSCs. The miCD34+ HSCs of the present invention exhibit limited self-renewal capacity but the potential for rapid pluripotency, which implies differentiation of PB-IPCs into CD34+ HSC-like cells through mitochondrial reprogramming, expression of HSC-associated cell surface markers, and primarily improved pluripotency, without significantly altering their self-renewal capacity.

[0032] III. Materials and Methods for Generating Pluripotent Stem Cells from miPB-IPCs A.PB-IPC cell culture

[0033] Human buffy coat blood units (n = 42; mean age 47.64 ± 14.07; age range 16-73 years; 23 males and 19 females) were purchased from the New York Blood Center (New York, NY, USA, http: / / nybloodcenter.org / ). Human buffy coats were first added to 40 mL of chemically defined, serum-free culture X-VIVO™ 15 medium (Lonza, Walkersville, MD, USA), thoroughly mixed with a 10 mL pipette, and then used for the isolation of peripheral blood-derived mononuclear cells (PBMCs). PBMCs were collected as previously described (Zhao et al., "A human peripheral blood monocyte-derived subset acts as pluripotent stem cells," Proc. Natl. Acad. Sci. USA 2003, 100, 2426-2431, incorporated herein by reference in its entirety). Mononuclear cells were isolated from buffy-coat blood using Ficoll-Paque™ PLUS (γ = 1.007, GE Healthcare) followed by red blood cell removal using Red Blood Cell Lysis buffer (eBioscience, San Diego, CA, USA). After three washes with saline, whole PBMCs were cultured at 1 x 10 per 150 x 15 mm Petri dish (BD, Franklin Lakes, NJ, USA) in chemically defined, serum-free culture medium X-VIVO™ 15 (Lonza, Walkersville, MD, USA) without the addition of any other growth factors. 6Cells were seeded at 25 mL / dish and incubated at 37°C and 8% CO2. After 7 days, peripheral blood insulin-producing cells (PB-IPCs) grew and expanded by adhering to the hydrophobic bottom of the Petri dish. Subsequently, PB-IPCs were washed three times with saline to remove all floating cells. Serum-free NutriStem® hPSC XF medium (Corning, New York, NY, USA) was added, and continued cell culture and expansion were performed at 37°C and 8% CO2. Expanded PB-IPCs were typically used for experiments for 7 to 14 days. PB-IPCs were treated with 100 μg / mL platelet-derived mitochondria for 7 to 14 days in untreated 24-well plates or Petri dishes containing serum-free NutriStem® hPSC XF culture medium (Corning) at 37°C and 8% CO2.

[0034] B. Isolation of Mitochondria from Platelets Mitochondria were isolated from peripheral blood (PB) platelets using a mitochondrial isolation kit (Thermo Scientific, Rockford, IL, USA, Prod: 89874) according to the manufacturer's recommended protocol. Adult human platelet units (n = 19) were purchased from the New York Blood Center (New York, NY, USA, http: / / nybloodcenter.org / ). Mitochondrial concentration was determined by measuring protein concentration using a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). Isolated mitochondria were aliquoted and stored in a -80°C freezer in preparation for experiments.

[0035] For fluorescent mitochondrial staining, mitochondria were labeled with MitoTracker Deep Red FM (100 nM) (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C for 15 min according to the manufacturer's recommended protocol, followed by washing twice with phosphate-buffered saline (PBS) at 3000 rpm for 15 min.

[0036] C. Flow Cytometry Flow cytometry analysis of surface and intracellular markers was performed. PB-IPCs were washed with PBS at 2000 rpm for 5 min. Mitochondria were washed with PBS at 12,000 g for 10 min at 4°C. PB-IPC nuclei were washed with PBS at 500 g for 5 min at 4°C. Samples were preincubated with human BD Fc block (BD Pharmingen, San Jose, CA, USA) for 15 min at room temperature and then directly divided into aliquots for staining with different antibodies. The cells were incubated with different mouse anti-human monoclonal antibodies (mAbs) from Beckman Coulter (Brea, CA, USA), including FITC-conjugated anti-CD45RO, anti-CD19, anti-CD4, anti-CD8, and anti-CD42a; ​​phycoerythrin (PE)-conjugated anti-CD34, anti-CCR7, and anti-CXCR4; phycoerythrin-Cy5.5 (PE-Cy5.5)-conjugated anti-CD19, anti-CD117, and anti-SOX2; phycoerythrin-Cy7 (PE-Cy7)-conjugated anti-CD41, anti-CD11b, and anti-CD45; APC-conjugated anti-CD34, anti-CXCR4, and anti-CD4; APC-Alexa Fluor 750-conjugated anti-CD66b and anti-CD8; Pacific Blue (PB)-conjugated anti-CD38; and Chrome Orange-conjugated anti-CD14. FITC-conjugated anti-human lineage cocktail 1 (Lin1) (CD3, CD14, CD16, CD19, CD20, CD56), Alexa Fluor 488-Sox2, Alexa Fluor 647-conjugated mouse anti-human C-peptide, and insulin antibodies were purchased from BD Biosciences (San Jose, CA, USA). FITC-conjugated anti-human MAFA antibody was purchased from United States Biological (Salem, MA, USA). APC-conjugated mouse anti-human CD36 mAb was purchased from BioLegend (San Diego, CA, USA). PE-conjugated anti-human GLUT2 antibody was purchased from R&D Systems (Minneapolis, MN, USA). Mouse anti-SDF-1 polyclonal antibody was purchased from Abcam (Cambridge, MA, USA).eFluor 660-conjugated rat anti-human OCT3 / 4 and isotype-matched IgG antibodies were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

[0037] For surface staining, cells were stained for 30 minutes at room temperature and then washed with PBS at 2000 rpm for 5 minutes before flow analysis. An isotype-matched mouse anti-human IgG antibody (Beckman Coulter) served as a negative control for all fluorescein-conjugated IgG mAbs. For intracellular staining, cells were fixed and permeabilized according to the manufacturer's recommended protocol using the PerFix-nc kit (Beckman Coulter). After staining, cells were collected and analyzed using a Gallios Flow Cytometer (Beckman Coulter) equipped with three lasers (488 nm blue, 638 nm red, and 405 nm violet laser) for simultaneous reading of up to 10 colors. Final data were analyzed using Kaluza Flow Cytometry Analysis software (Kaluza Analysis 2.1, Beckman Coulter).

[0038] To determine insulin-producing cells in mouse peripheral blood, MIP-GFP transgenic mice were tested according to an animal protocol approved by the Institutional Animal Care and Use Committee (IACUC). MIP-GFP transgenic mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). The strain was B6.Cg-Tg(Ins1-EGFP)1Hara / J (strain number: 006864).

[0039] D. Differentiation of miPB-IPCs into retinal pigment epithelial (RPE) cells To assess the pluripotency and retinal pigment epithelial (RPE) cell differentiation of mitochondria-induced PB-IPCs (miPB-IPCs) (Figures 1 and 43), miPB-IPCs were treated with a complex supplement (containing L-glutamine, gentamicin sulfate-amphotericin (GA-1000), and basic fibroblast growth factor) in 24-well tissue culture-treated plates in the presence of retinal pigment epithelial growth medium (Lonza) at 37°C and 5% CO for 8 days. Differentiated cells were characterized by immunocytochemistry using RPE-specific markers, such as mouse anti-human mAb RPE65, CRALBP, and claudin-19, and rabbit anti-tight junction protein 1 (ZO-1) polyclonal antibody (Novus Biological, Littleton, CO, USA). Human primary RPE cells were purchased from Lonza and used as a positive control. Isotype-matched IgG was used as a negative control for immunostaining. For functional analysis, differentiated RPE cells were subjected to phagocytosis of fluorescent latex beads (Sigma, Saint Louis, MO, USA). The phagocytosis-associated surface marker CD36 was examined by flow cytometry. CD36 expression levels were quantified by mean fluorescence intensity after analysis with Kaluza software version 2.1 (Beckman Coulter).

[0040] E. Neuronal differentiation of miPB-IPCs To determine the pluripotency of miPB-IPCs and their neuronal differentiation (Figures 1 and 43), miPB-IPCs were treated with 100 ng / mL neuron growth factor (NGF, R&D Systems) and human neuronal stem cell growth medium (iXCells Biotechnologies, San Diego, CA, USA) in 24-well tissue culture-treated plates at 37°C and 5% CO for 3–5 days. Differentiated cells were characterized by immunocytochemistry using mouse anti-human tyrosine hydroxylase monoclonal antibody (mAb, clone LNC1, catalog #MAB318, 1:100 dilution) and rabbit anti-synapsin I polyclonal antibody (catalog #AB1543, 1:100 dilution) (EMD Millipore, Temecula, CA, USA). FITC-conjugated AffiniPure donkey anti-mouse secondary antibody and Cy3-conjugated AffiniPure donkey anti-rabbit secondary antibody were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Isotype-matched IgG was used as a negative control for immunostaining. After covering with mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, USA), cells were photographed using a Nikon A1R confocal microscope on a Nikon Eclipse Ti2 inverted stage.

[0041] F. Colony Analysis miPB-IPCs were initially cultured in serum-free NutriStem® hPSC XF Medium (Corning) at 1x10 cells / well in a 24-well tissue culture plate. 4The miPB-IPCs were cultured at 37°C and 8% CO2 at 2000 cells / mL / well. After 2 months of culture, individual colonies were harvested under an inverted microscope using a BD Vacutainer blood collection set (21G 3 / 4" 12") attached to a 3 mL syringe (Nipro, Miami, FL, USA) and inoculated into a 96-well plate. Light microscopic examination showed that approximately 80% of the wells contained a single colony. Wells containing multiple colonies were excluded. Each single colony (n = 21 colonies in total) was manually dispersed by pipetting and aliquoted into 2 to 8 wells (depending on colony size) for differentiation induction (Figures 1 and 4). Differentiation of single-colony-derived cells into different lineages was investigated using the conditions described above.

[0042] For macrophage differentiation, cells derived from three colonies were treated with M-CSF and HSC-Brew GMP basal medium for 2–3 days. For RPE cell differentiation, cells derived from six colonies were treated with the RtEGM™ Retinal Pigment Epithelial Cell Growth Medium Kit™ (Lonza) for 3–5 days. For neuronal differentiation, cells derived from a single colony (total n = 9 colonies) were treated with 100 ng / mL nerve growth factor (NGF, R&D Systems) and human neural stem cell growth medium (iXCells Biotechnologies, San Diego, CA, USA) for 2–3 days in a 96-well plate. Their differentiation was assessed by lineage-specific markers, including phagocytosis of fluorescent latex beads for macrophage differentiation, RPE65 immunostaining for RPE cells, and synapsin I immunostaining for neurons. Untreated colony-derived cells were used as controls. To further determine their phenotype, single colony-derived cells (n = 3 colonies) were examined by flow cytometry using the leukocyte common antigen CD45, the HSC marker CD34, the ES cell marker SOX2, and the memory cell markers CD45RO and CCR7. Isotype-matched IgG was used as a flow cytometry control.

[0043] To determine the pluripotent differentiation of miPB-IPCs, initial colony analysis was performed using three germ layer-associated markers: the neuronal marker synapsin for ectoderm, the pancreatic islet β cell marker insulin for endoderm, and the macrophage marker CD11b for mesoderm. Further colony analysis was performed using the Human Definitive Pancreatic Endoderm Analysis Kit (Human Definitive Pancreatic Endoderm) with additional three germ layer-associated markers, including the neuronal marker βIII tubulin (Tuj1) for ectoderm, the hepatocyte marker α-fetoprotein (AFP) for endoderm, and the smooth muscle actin (SMA) for mesoderm, using a Tri-Germ Immunocytochemistry Kit (Invitrogen, Carlsbad, CA, USA). For immunostaining, colonies were fixed, permeabilized in 24-well plates, and then immunostained as described above. IgG was used as a negative control.

[0044] G. Tumorigenesis Assay To examine the tumorigenic potential of miPB-IPC, miPB-IPC were inoculated subcutaneously into the right flank of NSG mice (2 × 10 7 Cells / mouse, 200 μL saline, SC, right lower flank, n=3 mice). An equal volume of saline was injected into the left lower flank as a control. Tumor formation and body weight were monitored weekly for 12 weeks. At the end of the observation period, liver, lung, spleen, and kidney tissues of miPB-IPC-treated mice were examined and collected for histopathological examination for tumor formation.

[0045] H. Tracking RFP-labeled mitochondria in PB-IPCs To directly examine the penetration of red fluorescent protein (RFP)-labeled mitochondria into PB-IPCs, RFP-labeled mitochondria were purified from HEK-293 cells after labeling with CellLight™ Mitochondria-RFP BacMam2.0 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's recommended protocol. PB-IPCs were initially seeded onto 12 mm Nunc Glass Base Dishes (Thermo Fisher Scientific) in NutriStem™ hPSC XF culture medium. After 1 hour of attachment, PB-IPCs were treated with purified RFP-labeled mitochondria in X-VIVO™ 15 medium (Lonza). After 4 hours of treatment, the treated PB-IPCs were photographed using a confocal microscope. Hoechst 33,342 was applied to stain the nuclei of viable cells.

[0046] I. Transmission Electron Microscopy (TEM) To measure mitochondrial penetration into the nucleus, PB-IPCs were treated with 100 μg / mL platelet-derived mitochondria for 12 hours at 37°C and 8% CO2. Mitochondria-treated and untreated PB-IPCs were harvested at 500g for 5 minutes and fixed in 0.1M cacodylate buffer containing 2.5% glutaraldehyde / 4% paraformaldehyde for transmission electron microscopy (Philips CM12 microscope equipped with an AMT-XR11 digital camera). Alternatively, purified viable nuclei were labeled with Hoechst 33,342 and incubated with MitoTracker Deep Red-labeled mitochondria. These interactions were directly observed and photographed under a confocal microscope.

[0047] J. Blocking experiment using CXCR4 receptor antagonist AMD3100 To determine whether the action of SDF-1 / CXCR4 contributed to mitochondrial penetration into the nucleus, we performed a blocking experiment using the CXCR4 receptor antagonist AMD3100. Purified PB-IPC nuclei were treated with purified mitochondria labeled with MitoTracker Deep Red, with or without AMD3100 (30 μM). An equivalent concentration of the solvent, DMSO, was used as a control. After 4 h of treatment, nuclei were washed twice with PBS and prepared for flow cytometry.

[0048] K. Quantitative real-time PCR To clarify the interaction between mitochondria and nuclei, PB-IPC nuclei were isolated using a nuclear isolation kit (Sigma) according to the manufacturer's recommended protocol. PB-IPC nuclei were treated with 100 μg / mL platelet-derived mitochondria at 37°C and 8% CO for 4 hours. Mitochondria-treated and untreated nuclei were collected at 500g for 5 minutes and fixed in 2.5% glutaraldehyde / 4% paraformaldehyde in 0.1M cacodylate buffer for electron microscopy. Also, nuclei were collected at RT. 2 We applied the Profiler Real-Time PCR Array and used the Human Epigenetic Chromatin Modification Enzymes Kit (96-well format, Qiagen, Valencia, CA, USA) to study direct genetic and epigenetic regulation of mitochondria. 2 The Profiler Real-Time PCR Array was used according to the manufacturer's instructions, and data were analyzed using PrimePCR Array Analysis Software (Bio-Rad, Hercules, CA, USA).

[0049] To detect the expression of human islet β-related gene markers by quantitative real-time PCR, PB-IPCs were isolated from the cultured vessels after attachment for different time points, including 6, 12, 24, 48, and 72 hours. Total RNA was extracted from each sample using a Qiagen kit (Valencia, CA, USA). First-strand cDNA was synthesized from the total RNA using the iScript gDNA Clear cDNA synthesis kit according to the manufacturer's instructions (Bio-Rad, Hercules, CA, USA). Real-time PCR was performed on each sample in triplicate using a StepOnePlus Real-Time PCR System (Applied Biosystems, CA, USA) under the following conditions: 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. Validated gene-specific PCR primer sets were used for each gene, including islet cell-associated markers such as insulin (Bio-Rad Laboratories, Hercules, CA, USA), MAFA, NKX6.1, and PDX-1 (Qiagen, Valencia, CA, USA). The expression level of each gene was determined relative to β-actin as an internal control. To confirm gene expression, real-time PCR products were examined by 1.5% agarose gel electrophoresis.

[0050] L.RNA sequencing (RNA-seq) RNA sequencing (RNA-seq) analysis was performed between mitochondria-treated and untreated PB-IPCs in four preparations. Total RNA from each sample was extracted using a Qiagen kit (Valencia, CA, USA) and shipped on dry ice to Genewiz (South Plainfield, NJ, USA) for standard RNA sequencing and profiling of gene expression using a 2x150bp per lane, single-index Illumina NovaSeq™ 6000 sequencing system (Genewiz, South Plainfield, NJ, USA).

[0051] M. Statistics information Statistical analysis of the data was performed by two-tailed paired Student's t-test to determine statistical significance between untreated and treated groups. Values ​​are expressed as mean ± SD (standard deviation).

[0052] IV. Materials and Methods for Generating Hematopoietic-Like Stem Cells from miPB-IPCs A.PB-IPC cell culture Human buffy coat blood units (n = 51; mean age 48.97 ± 14.11; age range 18-72 years; 24 males and 27 females) were purchased from the New York Blood Center (New York, NY, USA, http: / / nybloodcenter.org / ). Human buffy coats were first added to 40 mL of chemically defined, serum-free culture X-VIVO™ 15 medium (Lonza, Walkersville, MD, USA), thoroughly mixed with a 10 mL pipette, and then used for the isolation of peripheral blood-derived mononuclear cells (PBMCs). PBMCs were then harvested. Mononuclear cells were isolated from the buffy coat blood using Ficoll-Paque™ PLUS (γ = 1.007, GE Healthcare, Chicago, IL, USA), followed by red blood cell removal using red blood cell lysis buffer (eBioscience, San Diego, CA, USA). After washing three times with saline, whole PBMCs were cultured at 1x10 per 150x15mm Petri dish (BD Falcon, NC, USA) in chemically defined serum-free culture X-VIVO™ 15 medium (Lonza, Walkersville, MD, USA) without the addition of any other growth factors. 6 Cells / mL, 25 mL / dish, were seeded and incubated at 37°C, 8% CO2. After 7 days, PB-IPCs expanded by growing and adhering to the hydrophobic bottom of the Petri dish. PB-IPCs were then washed three times with saline to remove all floating cells. Serum-free NutriStem® hPSC XF Medium (Corning) was then added, and cell culture and expansion continued at 37°C, 8% CO2. Expanded PB-IPCs were used for experiments within 7–14 days.

[0053] B. Isolation of Mitochondria from Platelets Mitochondria were isolated from peripheral blood (PB) platelets using a mitochondrial isolation kit (Thermo Scientific, Rockford, IL, USA, Prod: 89874) according to the manufacturer's recommended protocol. Adult human platelet units (n = 16; mean age 30.81 ± 8.64; age range 16-40 years; 9 males and 7 females) were purchased from the New York Blood Center (New York, NY, USA, http: / / nybloodcenter.org / ). Mitochondrial concentration was determined by measuring protein concentration using a NanoDrop 2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). Isolated mitochondria were aliquoted and stored in a -80°C freezer for further experiments.

[0054] For fluorescent mitochondrial staining, mitochondria were labeled with MitoTracker Deep Red FM (100 nM) (Thermo Fisher Scientific, Waltham, MA, USA) at 37°C for 15 min according to the manufacturer's recommended protocol, followed by washing twice with PBS at 3000 rpm for 15 min.

[0055] C. In vitro differentiation of PB-IPCs into miCD34+ HSCs PB-IPCs were treated with 100 μg / mL platelet-derived mitochondria for 7–14 days at 37°C and 8% CO2 in untreated 24-well plates or Petri dishes containing serum-free NutriStem™ hPSC XF culture medium (Corning, New York, NY, USA). According to the current protocol, mitochondrial-induced CD34+ hematopoietic-like stem cells (miCD34 +miCD34 HSCs were purified from mitochondria-treated PB-IPCs by immunomagnetic sorting using the Miltenyi Biotech CD34 MicroBead Kit (Miltenyi Biotech, Gladbach, Germany, catalog #130-097-047) according to the manufacturer's instructions. + HSC differentiation was characterized by flow cytometry.

[0056] D. Flow Cytometry Flow cytometry analysis of surface and intracellular markers was performed. PB-IPCs were washed with PBS at 2000 rpm for 5 minutes. Mitochondria were washed with PBS at 12,000 g for 10 minutes at 4°C. Samples were preincubated with human BDFc block (BD Pharmingen, Franklin Lakes, NJ, USA) for 15 minutes at room temperature and then directly aliquoted for staining with different antibodies. Cells were incubated with different mouse anti-human monoclonal antibodies (mAbs). For surface staining, cells were stained for 30 minutes at room temperature and then washed with PBS at 2000 rpm for 5 minutes before flow analysis. An isotype-matched mouse anti-human IgG antibody (Beckman Coulter, Brea, CA, USA) was used as a negative control for all fluorescein-conjugated IgG mAbs. Nucleated red blood cells were determined using SYTO™ 60 (Thermo Fisher, Waltham, MA, USA) in combination with CD235a (GLY-A) staining. Staining with propidium iodide (PI) (BD Biosciences, San Jose, CA, USA) was used to exclude dead cells during flow cytometry analysis. For intracellular staining, cells were fixed and permeabilized according to the manufacturer's recommended protocol using the PerFix-nc kit (Beckman Coulter). After staining, cells were collected and analyzed using a Gallios Flow Cytometer (Beckman Coulter, Brea, CA, USA) equipped with three lasers (488 nm blue, 638 nm red, and 405 nm violet laser) for simultaneous reading of up to 10 colors. Final data were analyzed using Kaluza Flow Cytometry Analysis Software version 2.1 (Beckman Coulter).

[0057] The cells were immunoblotted with different mouse anti-human monoclonal antibodies (mAbs) from Beckman Coulter (Brea, CA, USA), such as FITC-conjugated anti-CD45RA, anti-IFNγ, anti-CD4, anti-CD235a, anti-CD8, and anti-CD42a; ​​phycoerythrin (PE)-conjugated anti-CD34; PE-Texas Red-conjugated CD3; phycoerythrin-Cy5 (PE-Cy5)-conjugated anti-CD90; phycoerythrin-Cy5.5 (PE-Cy5.5)-conjugated anti-CD19; phycoerythrin-Cy7 (PE-Cy7)-conjugated anti-CD49f, anti-CD11b, and anti-CD45; APC-conjugated anti-CD4; APC-Alexa Fluor 700-conjugated anti-CD71; and APC-Alexa Fluor 700-conjugated anti-CD49f. The cells were incubated with BV510-conjugated anti-CD7, anti-CD66b, and anti-CD8; Pacific Blue (PB)-conjugated anti-CD38; Krome Orange-conjugated anti-CD14 and anti-CD135 (FLT3)-conjugated anti-CD45; AlexaFluor-488-conjugated anti-human cytochrome C; FITC-conjugated anti-CD90 (THY1) and anti-CD11C; BV510-conjugated anti-CD45; PE-conjugated anti-IL4, anti-IL5, anti-BAH1, and anti-IL12; PE-CF594-conjugated anti-CD10; BV421-conjugated anti-CD209; and PE-conjugated anti-mouse CD45.1, all from BD Biosciences (San Jose, CA, USA). Antibodies such as FITC-conjugated anti-human Hsp60, anti-human TCRαβ, anti-human Notch1, and anti-human Notch2; PE-conjugated anti-human Notch3, anti-human Dll1, anti-human Dll4, and anti-human Jagged2; APC-conjugated anti-human Notch4; phycoerythrin-Cy7 (PE-Cy7)-conjugated anti-TCRγδ, and Pacific blue (PB)-conjugated anti-CD3 were purchased from Biolegend (San Diego, CA, USA). FITC-conjugated anti-human Jagged1 and PE-conjugated anti-human Dll3 mAbs were purchased from R&D Systems (Minneapolis, MN, USA).Hemoglobin β / γ / δ (H-76) rabbit polyclonal antibody, AlexaFluor-546-conjugated calnexin, and AlexaFluor-647-conjugated GM130 were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). SYTO™ 60 was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

[0058] E. Multiple differentiation of miCD34+ HSCs First, miCD34 was isolated from miPB-IPCs using a Human Lyophilized CD34 MicroBead Kit (Miltenyi Biotec, Gladbach, Germany) via an automated MACS Pro Separator (Miltenyi Biotec, Gladbach, Germany) according to the manufacturer's recommended protocol. + HSCs were purified. Purified miCD34 + HSCs were treated with various inducers for cell differentiation.

[0059] Purified miCD34 to test T cell differentiation + HSC(1x10 5 Cells (1000 cells / mL) were seeded into 24-well untreated plates in the presence of HSC-Brew GMP basal medium (Miltenyi Biotec, Gladbach, Germany) supplemented with the cytokines 25 ng / mL hFlt3L and 25 ng / mL rhIL-7 (R&D Systems, Minneapolis, MN, USA) at 37°C and 5% CO2. After 3–7 days of treatment, cells were imaged and analyzed by confocal microscopy and flow cytometry using different T cell markers, including CD3, CD4, CD8, TCRα / β, CD38, Th1 cytokines (IL-4 and IL-5), and Th2 cytokines (IFN-γ and IL-12). Untreated miCD34 +HSCs were used as a negative control. T cells from healthy donors were used as a positive control. For combined immunocytochemistry, differentiated cells were fixed in 24-well plates with 4% paraformaldehyde for 20 min, permeabilized with 0.5% Triton X-100 (Sigma, Saint Louis, MO, USA) for 5 min, and blocked nonspecific binding with 2.5% horse serum. Subsequently, they were immunostained with FITC-conjugated mouse anti-human CD4 and CD8 (Beckman Coulter, Brea, CA, USA). After covering with mounting medium containing DAPI (Vector Laboratories, Burlingame, CA, USA), cells were photographed with a Nikon A1R confocal microscope on a Nikon Eclipse Ti2 inverted stage using NIS Elements version 4.60 software.

[0060] miCD34 + Purified miCD34 for differentiation of HSCs into macrophages + HSC(1x10 5 MiCD34 (cells / mL) were treated with 50 ng / mL M-CSF (Sigma, St. Louis, MO, USA) in 24-well untreated plates at 37°C and 5% CO2 in the presence of HSC-Brew GMP basal medium. After 2-3 days of treatment, cells were analyzed by flow cytometry using the phagocytic and macrophage markers CD11b (Beckman Coulter, Brea, CA, USA) and CD209 (BD Biosciences, San Jose, CA, USA). Untreated miCD34 + HSCs were used as a negative control. To detect the function of differentiated macrophages, fluorescent latex beads (Sigma, Saint Louis, MO, USA) were immobilized on the miCD34 cells of M-CSF-treated and untreated HSCs. + The beads were added to HSC cultures. After 4 hours of incubation with the latex beads, the cells were washed three times with PBS. Phagocytosis was observed and evaluated under a microscope. Positive cells had a minimum of five beads per cell.

[0061] miCD34 +To differentiate HSCs into granulocytes, purified miCD34 + HSC(1x10 5 Cells (1000 cells / mL) were treated with 25 ng / mL hFlt3L and 100 ng / mL G-CSF (R&D Systems) in 24-well untreated plates at 37°C and 5% CO2 in the presence of HSC-Brew GMP basal medium. After 3–5 days of treatment, cells were imaged and analyzed by flow cytometry using the granulocyte marker CD66b and staining with Wright-Giemsa (Sigma, Saint Louis, MO, USA) according to the manufacturer's instructions. Untreated miCD34 + HSCs were used as a negative control, and PBMCs from a healthy donor were used as a positive control.

[0062] miCD34 + To differentiate HSCs into RBCs, purified miCD34 + HSC(1x10 5 Cells (1000 cells / mL) were treated with 25 ng / mL hFlt3L and 3 units / mL EPO (R&D Systems, Minneapolis, MN, USA) in 24-well untreated plates at 37°C and 5% CO2 in the presence of HSC-Brew GMP basal medium. After 5 days of treatment, cells were re-treated with 3 units / mL EPO for an additional 3–7 days. Cells were then photographed and analyzed by flow cytometry using the erythrocyte markers CD235a and hemoglobin. Untreated miCD34 +HSCs were used as a negative control. For intracellular flow cytometry, all floating cells were collected and centrifuged at 2700 g for 15 minutes. After blocking nonspecific binding with Fc Blocker (BD Biosciences, San Jose, CA, USA), cells were fixed and permeabilized using the PerFix-nc kit (Beckman Coulter, Brea, CA, USA) according to the manufacturer's recommended protocol. Next, cells were incubated with rabbit anti-human hemoglobin β / γ / δ polyclonal antibody (Santa Cruze, Dallas, TX, USA) at a 1:100 dilution for 30 minutes at room temperature, followed by a 15-minute wash with PBS at 2700 g. Next, cells were labeled with Cy5-conjugated AffiniPure donkey anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 30 min in combination with staining with mouse anti-human CD235a-FITC (Beckman Coulter, Brea, CA, USA) and CD45-PE-CY7 mAb, followed by flow cytometry analysis.

[0063] miCD34 + Purified miCD34 was used to differentiate HSCs into megakaryocytes and platelets. + HSC(1x10 5 Cells (1000 cells / mL) were treated with 25 ng / mL hFlt3L and 100 ng / mL TPO (R&D Systems, Minneapolis, MN, USA) in 24-well untreated plates at 37°C and 5% CO2 in the presence of HSC-Brew GMP basal medium. After 3-7 days of treatment, cells were photographed and collected for flow cytometry using the MK / platelet marker CD42a (Beckman Coulter, Brea, CA, USA). Untreated miCD34 + HSCs were used as a negative control. For analysis of polyploidization, viable TPO-treated miCD34 +Cells were first stained with CD42a mAb and Hoechst 33342 (Sigma, Saint Louis, MO, USA) and imaged by confocal microscopy. Second, mature T cells (1N) and platelets (0N) from healthy donors were used as controls and were stained with propidium iodide (PI) (Abcam, Cambridge, MA, USA) according to the manufacturer's recommended protocol. + The ploidy of MK was analyzed by flow cytometry.

[0064] miCD34 + To determine morphological differentiation of HSCs into granulocytes, RBCs, and megakaryocytes / platelets, Wright-Giemsa staining was performed on treated and untreated cells, which were then observed and imaged using a Nikon ECLIPSE Ti2 inverted microscope.

[0065] For DAPT blocking experiments, PB-IPCs were treated with 100 μg / mL mitochondria and 10 μM DAPT (Sigma, Saint Louis, MO, USA, catalog #D5942) in non-tissue culture-treated 24-well plates or Petri dishes containing serum-free NutriStem® hPSC XF culture medium (Corning, New York, NY, USA) at 37°C and 8% CO2 for 7–14 days. Both treated and untreated PB-IPCs were then collected and examined for CD34 expression by flow cytometry.

[0066] F. Animal studies and engraftment of miCD34+ HSCs into irradiated NSG mice All animal experiments were performed in accordance with the approval of the Institutional Animal Care and Use Committee at Hackensack Meridian Health. + To demonstrate the pluripotent properties of HSCs, purified miCD34 + Irradiated NOD / Lt-scid / IL2Rγ HSCs nullNSG mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and bred and maintained under pathogen-free conditions in the animal facility at the Center for Discovery and Innovation.

[0067] miCD34 + To determine the repopulation potential of HSCs, 12- to 16-week-old NSG mice were irradiated with 200 cGy using an RS-2000 irradiator (Rad Source Technologies, Suwanee, GA, USA). 24 hours after irradiation, 3 x 10 mice were injected via the tail vein (200 μL saline, n = 10 mice) according to a protocol approved by the Animal Care and Use Committee (ACC) at Hackensack Meridian Health. 5 miCD34 in cells / mouse + HSCs were transplanted into irradiated NSG mice. Saline injection (200 μL) alone was used as a control (n=6 mice). After transplantation, mice were observed twice a week for 16 weeks. miCD34 + To examine HSC differentiation, mice were sacrificed at 12 or 16 weeks after transplantation, and peripheral blood, spleen, and bone marrow samples were collected for flow cytometry analysis. + To determine the multilineage differentiation of HSCs, only live cells from different samples were gated for analysis after excluding propidium iodide (PI)-positive dead cells. The gated live cells, which were human leukocyte common antigen (CD45)-positive and mouse CD45.1-negative, were further analyzed to characterize them using various human blood lineage-specific surface markers, including CD3 and CD4 for T cells; CD19 for B cells; CD41b for megakaryocytes / platelets; CD14, CD11b, and CD11c for monocytes / macrophages; CD66b for granulocytes; and CD235a for erythrocytes. SYTO60 is a marker for CD235a. + Used to stain nucleated erythroid cells. Determine T cell populations and CD4 +In addition to accounting for differences in cell size, anti-CD3 antibodies were used to remove monocytes, as well as CD4 + Monocytes were gated, and isotype-matched IgG was used as a flow cytometry control.

[0068] G. Statistics information Statistical analysis was performed using GraphPad Prism8 (version 8.0.1) software. Normality of samples was assessed using the Shapiro-Wilk test. Statistical analysis of data was performed using a two-tailed paired Student's t-test to determine statistical significance between untreated and treated groups. For nonparametric data, the Mann-Whitney U test was used. Values ​​are presented as mean ± SD (standard deviation). Statistical significance was defined as p < 0.05, two-tailed.

[0069] Although particular embodiments and / or aspects of the present invention have been shown and described, it should be understood that the present invention is not limited thereto and encompasses various other embodiments and aspects. [Example]

[0070] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. To the extent that specific materials are mentioned, this is for illustrative purposes only and is not intended to limit the invention. One skilled in the art of the invention may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

[0071] Example 1 Adult peripheral blood-derived PB-IPCs express human islet β cell-specific markers. Figures 3-10 show the characterization of peripheral blood-derived insulin-producing cells (PB-IPCs) derived from adult peripheral blood with pancreatic islet β cell-associated markers. To characterize the specific markers of PB-IPCs, PB-IPCs were purified from adult peripheral blood by their ability to adhere to the hydrophobic plastic surface of a Petri dish in serum-free medium. Flow cytometry revealed that PB-IPCs express insulin-producing cells in the presence of islet β cell-associated markers. - CD34 - CD45 + SOX2 + CD45RO + CCR7 + The PB-IPCs were demonstrated to display a phenotype (e.g., expression of the leukocyte common antigen CD45, memory cell markers CD45RO and CCR7, and embryonic stem (ES) cell marker SOX2) and had low expression of CD117 (Figure 3). In contrast, the PB-IPCs were negative for HSC markers CD34 and CD38; T cell markers CD3, CD4, and CD8; B cell marker CD19; granulocyte marker CD66b; and MK / platelet markers CD41 and CD42a (Figure 4). The CD14 markers, which were unable to adhere to the hydrophobic surface of the culture vessel, were also negative. + Monocytes / macrophages underwent apoptosis and / or necrosis within 24 hours of culture (Figure 5). Furthermore, we analyzed the cell cycle of freshly isolated PB-IPCs after overnight adherence by flow cytometry and propidium iodide (PI) staining. The data showed that 0.9 ± 0.5% of freshly isolated PB-IPCs were distributed in S phase, 92.94 ± 2.75% in G0 / G1 phase, and 6.68 ± 2.2% in G2 / M phase (Figure 6). This indicates that the cell proliferation potential of freshly isolated PB-IPCs is limited. Therefore, PB-IPCs exhibit a unique phenotype and are distinct from mesenchymal stem cells (MSCs) and monocyte-derived stem cells (termed fibroblast-like macrophages, f-Mφ).

[0072] Next, we analyzed insulin production in PB-IPCs. Real-time PCR data using human islet cells as a positive control revealed that PB-IPCs expressed human islet β cell-specific markers, including insulin and transcription factor (PDX-1, NKX6.1, and MAFA) mRNA (Figure 7). Kinetic analysis demonstrated that these gene markers remained stable in most PB-IPC samples within 24 hours of ex vivo culture in the presence of serum-free X-VIVO™ 15 medium, although some markers were lost or downregulated. This may be due to differences in the health status of blood donors. Flow cytometry further confirmed double-positive cells expressing MAFA and C-peptide (an insulin by-product) at the protein level (Figure 8). Because MAFA is the only pancreatic islet β cell-specific activator involved in insulin expression, and glucose transporter 2 (GLUT2) is a surface marker for human pancreatic islet β cells, we further combined the above PB-IPC markers with additional markers, ES cell-associated transcription factors octamer-binding protein 3 / 4 (OCT3 / 4) and SRY-box-containing gene 2 (SOX2), to analyze the proportion of PB-IPCs in human peripheral blood mononuclear cells (PBMCs) using MAFA and GLUT2. FITC-conjugated anti-human lineage cocktail 1 (Lin1) (CD3, CD14, CD16, CD19, CD20, CD56) was applied to exclude known cell lineages, such as T cells, monocytes / macrophages, granulocytes, B cells, and natural killer (NK) cells. Anti-human leukocyte common antigen CD45 mAb was used to remove contaminating red blood cells (RBCs) and platelets during data analysis. MAFA (transcription factor) and GLUT2 (beta cell surface marker) were used to determine the islet beta cell-associated phenotype in PB-IPC. Flow cytometry analysis revealed a Lin of 0.0045 ± 0.004. 1 -CD34 - CD45 + CD45RO + CCR7 + SOX2 + OCT3 / 4 + MAFA + Glut2 +We demonstrated that PB-IPC cells exist in freshly isolated human PBMCs using Ficoll-Paque. After overnight (12 hours) adherence selection, PB-IPC cells were isolated from PBMCs and transfected with Lin1. - CD34 - CD45 + CD45RO + CCR7 + SOX2 + OCT3 / 4 + MAFA + The same phenotype, accompanied by the expression of Glut2+, can be observed (Figure 9). GFP-positive insulin-producing cells were also found in the peripheral blood of insulin promoter-green fluorescent protein (GFP)-transgenic mice (strain name: B6.Cg-Tg(Ins1-EGFP)1Hara / J, strain number: 006864) (Figure 10). Thus, these data established the presence of PB-IPCs in peripheral blood that can be isolated by the approach of the present invention.

[0073] Example 2 Ex vivo differentiation of mitochondria-induced PB-IPCs (miPB-IPCs) into retinal pigment epithelial (RPE) cells Platelets are enucleated cells that do not contain human genomic DNA. Apheresis platelets were obtained from the New York Blood Center with high purity (CD41 + CD42 + Platelets >99%). The purity of the isolated mitochondria was ≥90%.

[0074] The retinal pigment epithelium (RPE) is a monolayer of cells that fundamentally supports visual function and photoreceptor integrity. Dysfunction and loss of RPE cells are the primary cause of age-related macular degeneration (AMD), leading to blindness. To determine whether miPB-IPCs are multipotent, we performed differentiation studies into RPE cells. Figures 11–14 show the differentiation of miPB-IPCs into retinal pigment epithelial (RPE) cells. Treatment with a complex supplement (e.g., L-glutamine, gentamicin sulfate-amphotericin (GA-1000), and basic fibroblast growth factor) for 8 days in the presence of RPE growth medium resulted in over 90% of miPB-IPCs acquiring an RPE phenotype. This RPE phenotype includes pigmented granules in the cytoplasm, numerous cell processes of varying lengths (Figure 11), and expression of the visual cycle protein RPE65 and cellular retinaldehyde-binding protein (CRALBP). Furthermore, similar to primary human RPE cells (Figure 12, top), the tight junction-associated membrane proteins claudin-19 and zonular occludens-1 (ZO-1) (Figure 12, bottom) were present. Functional analysis revealed strong phagocytosis of fluorescent beads (Figure 13) and upregulated expression of the phagocytic marker CD36 (Figure 14), similar to human RPE cells. Untreated cells, used as a control, failed to show these changes. These results indicated that differentiated RPE cells had acquired the phenotype of human RPE cells.

[0075] Example 3 Ex vivo differentiation of mitochondria-induced PB-IPCs (miPB-IPCs) into neuronal cells During induction of RPE cell differentiation, several elongated neuronal-like cells were observed. Therefore, we investigated the neuronal differentiation potential of miPB-IPCs. Figures 15–16 show the neuronal differentiation of miPB-IPCs. After treatment with 100 ng / mL nerve cell growth factor (NGF) and human neural stem cell growth medium for 2–3 days in 24-well plates, 99% of the treated miPB-IPCs exhibited typical neuronal morphology, including elongated axon-like processes with branches and formed intercellular networks via dendrites (Figure 15). Double immunostaining revealed that 99.1% of the treated cells expressed the neuron-specific marker synapsin I and tyrosine hydroxylase, the rate-limiting enzyme for the biosynthesis of catecholamines (e.g., dopamine and norepinephrine) (Figure 16). Untreated cells only showed spontaneous differentiation (<3%). These data demonstrate the potential of miPB-IPCs to differentiate into adrenergic neurons.

[0076] Example 4 Clonal analysis of miPB-IPCs To further determine the pluripotency of miPB-IPCs, clonal analysis was performed. Figures 17-23 show the clonal analysis of miPB-IPCs and the tumorigenicity of miPB-IPCs. MiPB-IPC colony formation was observed at different sizes (Figure 17), and the colony-forming potential of miPB-IPCs was significantly increased after mitochondria treatment compared to untreated PB-IPCs (Figure 18). MiPB-IPC colony formation occurred at various sizes over a 2-month culture period in 24-well plates. miPB-IPCs were initially cultured in serum-free NutriStem® hPSC XF Medium (Corning) at 1x10 cells / well in 24-well tissue culture plates. 4 The cells were cultured at 1000 cells / mL / well under the conditions of 37°C and 8% CO2. Data are presented as the mean ± SD from five preparations. Flow cytometry showed that these colonies expressed the PB-IPC marker CD45. + and CD34 - (94.7±4.29%, n=3), SOX2 + (77.38±13.34%), and CD45RO +and CCR7 + It was confirmed that the cells retained their normal morphology (92.4 ± 3.6%) (Figure 19). Five colonies were dispersed and seeded into 96-well plates. After treatment with different lineage-specific inducers, such as 50 ng / mL M-CSF for macrophage differentiation, 100 ng / mL NGF for neurons, and RPE cells using specific conditioned media, various lineage markers demonstrated that 62.05 ± 6.43% of differentiated Mφ cells exhibited phagocytosis of fluorescent beads (Figure 20, left), 75.6 ± 4.8% of differentiated RPE cells were RPE65 positive (Figure 20, center), and 94.8 ± 1.7% of differentiated neurons were synapsin I positive (Figure 20, right), each with characteristic morphology. Untreated cells showed minimal spontaneous differentiation (<5%). These data demonstrate that single colony-derived cells can give rise to various cell lineages, including macrophages, RPE cells, and neurons, confirming the multipotency of miPB-IPCs.

[0077] Additional research has shown that 2x10 7 After miPB-IPC transplantation at a dose of 100 cells / mouse (sc), no tumor formation was observed. The miPB-IPC-transplanted mice gained weight during a 12-week follow-up (Figure 21, n = 3 mice), and no obvious tumor formation was observed upon histological examination (lung, liver, spleen, and kidney), indicating the safety of miPB-IPC administration.

[0078] To assess the pluripotent differentiation of miPB-IPCs, colony analysis was performed using tri-germ-associated markers, including the neuronal marker synapsin for ectoderm, the pancreatic islet β cell marker insulin for endoderm, and the macrophage marker CD11b for mesoderm. Confocal microscopy demonstrated that more tri-germ-positive cells were distributed in miPB-IPC-derived colonies than in mitochondria-untreated PB-IPC-derived colonies (Figure 22). Using a tri-germ immunocytochemistry kit (Invitrogen), colony analysis was repeated with additional tri-germ-associated markers, such as the neuronal marker beta III tubulin (Tuj1) for ectoderm, the hepatocyte marker alpha-fetoprotein (AFP) for endoderm, and the smooth muscle actin (SMA) for mesoderm. The data confirmed spontaneously differentiated tri-germ-positive cells in miPB-IPC-derived colonies (Figure 23). The number of positive cells was very low or negative in mitochondria-untreated PB-IPC-derived colonies (Figure 23). Thus, the data demonstrated the pluripotency of miPB-IPCs.

[0079] Example 5 Mitochondrial penetration into the nuclei of PB-IPCs To investigate the effect of exogenous platelet-derived mitochondria on PB-IPCs, we observed mitochondria mitochondria migrating into the nuclei of PB-IPCs according to the treatment conditions using an electron microscope, as shown in Figures 24-31. Mitochondria were observed to have passed through the nuclear membrane (Figure 24), located inside the nuclear matrix, near the nucleolus (Figure 25), and had a shape similar to that of mitochondria in the cytoplasm (Figure 25, indicated by arrows). This remarkable phenomenon was not observed in untreated PB-IPCs (Figure 26). To further confirm the entry of exogenous mitochondria into the nuclei of PB-IPCs, PB-IPCs were treated with red fluorescent protein (RFP)-labeled mitochondria isolated from the HEK293 cell line (Figure 27). After 4 hours of treatment, RFP infiltrating into the cytoplasm was observed using a confocal microscope. +Mitochondria were identified (Figure 27). To directly visualize the interaction between mitochondria and the nucleus, freshly purified PB-IPC-derived nuclei were treated with isolated MitoTracker Red-labeled mitochondria. Confocal images revealed direct interaction between mitochondria and the nucleus, with some labeled mitochondria entering the nucleus (Figure 28). Based on observations by transmission electron microscopy (TEM) and flow cytometry after staining with mitochondrial markers, such as MitoTracker Deep Red staining, anti-cytochrome C, and anti-heat shock protein (HSP) 60 mAb, the frequency of intranuclear mitochondria was approximately 1-3%.

[0080] Next, we investigated the molecular mechanism underlying mitochondrial nuclear translocation. Flow cytometry revealed that nuclei expressed the chemokine receptor CXCR4 (a ligand for stromal cell-derived factor (SDF)-1) (Figure 29), and mitochondria expressed SDF-1 (Figure 30). To determine whether the action of SDF-1 / CXCR4 contributed to mitochondrial penetration into the nucleus, we performed a blocking experiment using the CXCR4 receptor antagonist AMD3100. Purified PB-IPC nuclei were treated with MitoTracker Deep Red-labeled purified mitochondria, with or without AMD3100. After 4 hours of treatment, flow cytometry showed that the percentage of MitoTracker Deep Red-positive nuclei was significantly reduced after treatment with AMD3100 (Figure 31). This indicates that mitochondria entered the nucleus via the chemoattractant interaction between SDF-1 and CXCR4.

[0081] Furthermore, we observed that PBMC-derived mitochondria (but not platelet-derived mitochondria) were also able to penetrate the nuclei of PB-IPCs with an incidence of 2.09 ± 0.87%. Figures 32–34 show a comparison of SDF-1 expression among platelet-, PBMC-, and PB-IPC-derived mitochondria. Flow cytometry revealed that PBMC-derived mitochondria showed similar SDF-1 expression levels as platelet-derived mitochondria but significantly higher than PB-IPC-derived mitochondria. PB-IPCs were treated with MitoTracker Red-labeled PBMC-derived mitochondria (100 μg / ml) at 37°C and 5% CO2. After 4–6 h of treatment, cells were observed and imaged using a Nikon A1R confocal microscope on a Nikon Eclipse Ti2 inverted stage with NIS Elements version 4.60 software (Figures 32–34). Together with the results of blocking purified PB-IPC nuclei with the CXCR4 receptor antagonist AMD3100, these data indicate that the SDF-1 / CXCR4 pathway contributes to the translocation of mitochondria to the nuclear membrane of PB-IPC, leading to the permeabilization of the PB-IPC nuclei.

[0082] Example 6 Genetic and epigenetic changes in PB-IPCs after treatment with mitochondria To investigate the mechanism by which mitochondria regulate nuclear expression, viable nuclei from purified PB-IPCs were treated with isolated mitochondria for 4 hours at 37°C and 5% CO2, and transcriptional changes were assessed by real-time PCR array. The data revealed significant changes in the following epigenetic chromatin-modifying enzyme-related genes: DNA methyltransferase 1 (DNMT1), histone acetyltransferases (activating transcription factor-2 (ATF2), lysine acetyltransferase 2B (KAT2B), KAT5, and KAT8), histone methyltransferases (coactivator-associated arginine methyltransferase 1 (CARM1), mixed lineage leukemia protein (MLL), MLL3, protein arginine methyltransferase 5 (PRMT5), and PRMT6), and histone methyltransferase activity-associated SET (Enhancer of Su(var), Zeste, and Trithorax) domain protein (ASH1L (absent)). , small molecule, or homeotic)-like (Drosophila), SET domain containing 1A (SETD1A), and SETD5), histone phosphorylation (aurora kinase B (AURKB), AURKC, p21 protein-activated kinase 1 (PAK1), and ribosomal protein S6 kinase polypeptide 3 (RPS6KA3)), histone ubiquitination (DAZ-interacting protein 3 (DZIP3) and ubiquitin-conjugating enzyme E2B (UBE2B)), DNA and histone demethylase methyl-CpG-binding domain protein 2 (MBD2), and histone deacetylases (histone deacetylase 3 (HDAC3), HDAC4, HDAC5, HDAC6, HDAC8, HDAC9, and HDAC11)) (Figure 35).

[0083] These data suggest that mitochondria penetrating the nucleus contribute to both epigenetic and genetic regulation, leading to reprogramming of PB-IPCs. To identify more differentially expressed genes, we performed RNA sequencing (RNA-seq) analysis on four preparations between mitochondria-treated and untreated PB-IPCs (Figure 36). The results showed that 37 genes were significantly upregulated (Figure 37, p<0.05) and 9 genes were downregulated (Figure 38, p<0.05) in mitochondria-treated PB-IPCs. No other genes (n=15,388, 99.7% of genes) were significantly altered in PB-IPCs after treatment with mitochondria.

[0084] Example 7 Mitochondrially derived CD34 in mitochondria-induced PB-IPC (miPB-IPC) after treatment with platelet-derived mitochondria + -HSC-like cells (miCD34 + Ex Vivo Differentiation into HSCs Highly purified apheresis platelets were obtained from the New York Blood Center for the following experiments (>99%, CD41 + CD42 + Figures 39-42 show the CD34 expression after treatment with platelet-derived mitochondria. + Differentiation of PB-IPCs into HSC-like cells is shown. To measure the purity of mitochondria isolated from platelets, different markers, including MitoTrack Deep Red staining, anti-cytochrome C, and anti-heat shock protein (HSP) antibodies, were applied by flow cytometry. 60 antibodies were used for mitochondrial markers, calnexin for the endoplasmic reticulum (ER), and GM130 for the Golgi apparatus. Flow cytometry showed that 99% of the isolated mitochondria were positive for MitoTrack Deep Red, HSP60, and cytochrome C, while approximately 5% were positive for cytochrome C. + Calnexin + Cells and 4% cytochrome C + GM130 +The presence of mitochondria was observed (Figure 39). The double-positive staining results may be due to the interaction and conjugation of mitochondria with the ER or Golgi apparatus, respectively. Flow cytometry analysis showed that the purity of the isolated mitochondria was ≥90% (Figure 39). Platelet-derived mitochondria purified from autologous or allogeneic peripheral blood were prepared and treated with expanded PB-IPCs isolated from adult donor blood samples at the New York Blood Center (n = 51; mean age 48.76 ± 14.97; age range, 18-72 years; 24 males and 27 females). Notably, the expression of the HSC marker CD34 was upregulated in PB-IPCs after treatment with mitochondria. Phenotypic analysis of miPB-IPCs after 2 weeks of mitochondria treatment was striking, in that CD34 expression on miPB-IPCs increased from 0.71% ± 0.25% to 14.8% ± 3.1% (p = 7.88 x 10 6 , n=5) (Figure 40). Using an optimized cell marker panel, mitochondrial-derived CD34 + (miCD34 + ) cells, CD34 + CD38 - / low CD45RA - CD49f + CD90 + Flt3 - / low CD7 + CD10 + CD71 + BAH1 - / low The phenotype of normal blood CD34 cells from unmobilized healthy donors was observed (14.8% ± 3.1%, n = 5) (Figure 41). + CD45RA - CD90 + Flt3 - / low CD7 + CD71 + miCD34 compared with HSCs (0.49% ± 0.19%, n = 4). + The cells were CD34 + CD45RA - CD90 + Flt3 - / low CD7 + CD71+ These mice expressed similar surface markers (15.3% ± 2.9%, n = 5, p < 0.01) but expressed higher levels of CD10 (a marker that defines human lymphoid progenitors) (99.4% ± 0.36% vs. 20.6% ± 3.1%, p < 0.01), CD49f (a general biomarker for most stem cell populations) (98.8% ± 1.3% vs. 15.4% ± 2.9%, p < 0.01), and lower levels of BAH-1 (a marker for human megakaryocytic erythroid progenitors) (0.51% ± 0.2% vs. 32.5% ± 3.9%, p < 0.01) (Figures 41 and 42). Data are consistent with those of miCD34 + Co-expression of CD7 and CD10 (surface markers of common lymphoid progenitor (CLP) cells) on HSCs leads to the development of miCD34 + This suggests that HSCs have a high potential to give rise to lymphocytes.

[0085] Example 8 miCD34 + Differentiation of HSCs into T cells Figures 43 to 50 show purified miCD34 + In vitro differentiation of HSCs into T cells. miCD34 + To determine whether the cells functioned as stem cells, they were purified from miPB-IPC and treated with different inducers (Figure 43). First, purified miCD34 + The cells were treated with recombinant FMS-like tyrosine kinase (FLT)-3 ligand, interleukin (IL)-2, and IL-7 for 3 days to examine their potential for differentiation into T cells. Phase-contrast microscopy revealed significant morphological changes, with differentiated T cells in the cytokine-treated group exhibiting numerous cell clusters, with some cells released into the supernatant (Figure 44, right). Cells in the control group exhibited a smooth surface and did not exhibit any morphological changes (Figure 44, left, and Figure 45). Confocal microscopy showed that differentiated cells strongly expressed the human T cell marker CD4 and weakly expressed CD8 (Figure 46). Flow cytometry revealed that miCD34 + CD3 in HSCs + CD4 + CD8 - CD38+ T cells (percentage 76.93% ± 3.21% (Fig. 47, n = 4), CD3 + CD4 + TCRαβ + Differentiation into T cells (82.65% ± 5.2%, n = 3) (Figure 48) was further confirmed. Intracellular staining with T cell functional markers showed that these T cells produced the Th1 cytokine IL-12 (65.3% ± 20.1%, n = 3) and the Th2 cytokines IL-4 (28.5% ± 9.99%, n = 3) and IL-5 (53.9% ± 11.2%, n = 3), with very low levels of interferon (IFN)-γ (0.61% ± 0.3%, n = 3) (Figure 49). Additional functional tests confirmed significantly upregulated expression levels of cytokines such as IL-4 (p=0.0025, n=3), IL-5 (p=0.0049, n=3), and IL-12 (p=0.037, n=3) after treatment with phorbol 12-myristate 13-acetate (PMA) and ionomycin. The level of IFN-γ showed no significant change (p=0.085, n=3). The data confirmed that differentiated T cells responded to PMA / ionomycin stimulation (Figure 50). Highly efficient and rapid differentiation of T cells involved the miCD34 + It was clearly demonstrated that HSCs were converted into functional and committed hematopoietic progenitor cells.

[0086] Example 9 miCD34 + Ex vivo differentiation of HSCs into other hematopoietic lineages Figures 51 to 57 show the miCD34 + To further explore the differentiation potential of HSCs, we investigated the effect of miCD34 + Treatment of HSCs with macrophage colony-stimulating factor (M-CSF) for 3 days rapidly resulted in well-spaced adherent cells. Functional analysis demonstrated that M-CSF-treated miCD34 + HSCs were confirmed to exhibit potent phagocytosis of fluorescent latex beads (Fig. 51, center), whereas untreated cells were largely negative for this effect (Fig. 51, left). Flow cytometry revealed that 34.3% ± 4.3% of the cells expressed CD11b+ CD209 + Macrophages (Mφ), approximately 53.66% ± 3.8% of which are CD11b + CD209 - These cells were confirmed to be macrophages (Mφ) (Figure 51, right). In contrast, untreated miCD34 + HSCs contain CD11b + CD209 - Only 15.29% ± 1.5% macrophages were present, and CD11b + CD209 + Macrophages accounted for 0.43% ± 0.12%.

[0087] Next, miCD34 + HSCs were induced with granulocyte colony-stimulating factor (G-CSF). After 3 days, 81.14% ± 3.7% of treated cells expressed the granulocyte-specific marker CD66b, and showed a reduced nuclear-cytoplasmic ratio and multilobed nuclei as shown by Wright-Giemsa staining (Figure 52). However, untreated miCD34 + HSCs failed to express CD66b and displayed large nuclei with a high nuclear-to-cytoplasmic ratio (Figure 52, n=4). Furthermore, after treatment with erythropoietin (EPO) for 5 days, miCD34 + HSCs became nucleated cells strongly positive for the erythroid (Er) lineage marker CD235a, and through elimination of the nucleus by additional EPO treatment, promoted RBC maturation, exhibiting the characteristic biconcave shape and enucleated RBCs (Figure 53). A total of 41.4% ± 11.46% of the cells were ultimately enucleated CD235a. + CD45 - hemoglobin + The mature RBCs differentiated into RBCs (Figure 54, n=4). However, untreated cells failed to demonstrate these differentiations or expressed only background levels of these markers. Furthermore, flow cytometry analysis revealed increased expression levels of hemoglobin in mature RBCs, and hemoglobin + CD45 - The mean fluorescence intensity of mature RBCs increased to 13.61 ± 4.29, and hemoglobin + CD45 +Immature RBCs were identified as 8.29 ± 1.61 (p = 0.044, n = 4).

[0088] In addition, miCD34 is important for MK and platelets, which are important for blood coagulation. + The involvement of HSCs was investigated. After 7 days of treatment with FLT-3 ligand and thrombopoietin (TPO), CD42 + MK production was obtained, with typical polyploidization (mostly 2N-7N) (Figures 55-57) and non-nucleated CD42 + Platelet formation (21.3% ± 4.1%, n = 4) (Figures 55-56) was achieved, yielding 95 ± 17 platelets per MK. Mature CD42 + Approximately 54% of the platelets were released into the supernatant (Fig. 3F, n = 4).

[0089] Example 10 miCD34 after transplantation into NSG mice + In vivo differentiation of HSCs into other hematopoietic lineages To further determine its pluripotency function, purified miCD34 + Irradiated HSCs in non-obese diabetic (NOD) / Lt-scid / IL2Rγ null The miCD34 expression level after transplantation into irradiated NSG mice was 100%. + Multiple in vivo differentiation of HSCs. Human CD45 expression in the peripheral blood, spleen, and bone marrow of miCD34 HSC-transplanted mice. + Cell chimerism was observed in T cells (CD3 + CD4 + ), B cells (CD19 + ), monocytes (CD14 + ), granulocytes (CD66b + ), erythrocytes (CD235a + ), and megakaryocytes / platelets (CD41b + The results were examined at 12 weeks using flow cytometry analysis with blood lineage-specific markers, including miCD34, after transplantation into irradiated NSG mice. +To determine the multilineage differentiation of HSCs, only live cells from different samples were gated for analysis after excluding propidium iodide (PI)-positive dead cells. Gated live cells, positive for human leukocyte common antigen (CD45) and negative for mouse CD45.1, were analyzed to characterize them using various lineage-specific surface markers, including CD3 and CD4 for T cells; CD19 for B cells; CD41b for megakaryocytes / platelets; CD14, CD11b, and CD11c for monocytes / macrophages; CD66b for granulocytes; and CD235a for erythrocytes. SYTO60 was used to stain CD235a+ nucleated erythroid cells. Isotype-matched IgG was used as a control for flow cytometry.

[0090] Human CD45 in the blood + Engraftment levels in cells (9.93% ± 9.62%, p = 0.035%) and spleen (25.37% ± 21.89%, p = 0.018) were much higher than in bone marrow (0.33% ± 0.15%) at 12 weeks post-engraftment (Figure 58, n = 6 mice). + HSC-derived CD3 + CD4 + T cells express human CD45 + The CD3+ / CD4+ / CD6+ / CD8+ / CD9+ / CD10+ / CD11+ / CD12+ / CD13+ / CD14+ / CD15+ / CD16+ / CD17+ / CD18+ / CD19+ / CD20+ / CD21+ / CD22+ / CD23+ / CD35+ / CD19+ / CD24+ / CD19+ / CD25+ / CD36+ / CD18+ / CD + CD4 + 59% ± 13.55% of T cells expressed miCD34 + It was obtained from the spleen cells of HSC-transplanted mice (Figures 59-60, n = 5 mice). In contrast, miCD34 in the bone marrow + HSC-derived CD3 + CD4 + The percentage of T cells was 49.45% ± 14.01% at 12 weeks, and CD19 + B cells were 5.24% ± 2.68%, CD41b + Megakaryocytes / platelets were 4.3% ± 2.0%, CD14 + Monocytes, 1.71% ± 2.36%, CD66b + Granulocytes, 0.51% ± 0.46%, CD235a + SYTO60 +Nucleated red blood cells were 0.22% ± 0.13%, CD235a + SYTO60 - The percentage of enucleated red blood cells was 0.08% ± 0.05% (Figure 61, n = 6 mice). + CD14 in the peripheral blood of HSC-transplanted mice + The percentage of monocytes was 2.1% ± 1.81%. Further flow cytometry showed that undifferentiated miCD34 + At 12 weeks, human CD34 HSCs were detected in the peripheral blood (0.07% ± 0.04%), spleen (0.04% ± 0.03%), and bone marrow (0.01% ± 0.01%) of mice transplanted with miCD34 HSCs. + Few cells were present (Figure 62, n=5 mice).

[0091] miCD34 induces monocyte / macrophage (myeloid differentiation) + To further confirm HSCs, miCD34 + Further animal studies were performed in HSC-transplanted mice at 12 and 16 weeks using anti-human CD11b and CD11cm Abs, respectively, for macrophage-associated markers. Flow cytometry revealed that miCD34 + mCD45 in splenocytes from HSC-transplanted mice at 16 weeks - hCD45 + hCD3 - hCD11b + hCD11c + The percentage of macrophages was shown to be 75.22% ± 18.33% (Figure 63, n = 3). In contrast, mCD45 - hCD45 + hCD3 + hCD11b - The percentage of T cells decreased from 59% ± 14.01% at 12 weeks to 4.05% ± 2.87% at 16 weeks (Figure 63). Therefore, the data suggest that the miD34 T cells were significantly reduced after transfer to irradiated NSG mice. + These data suggest that miCD34 may be involved in the differentiation of HSCs into monocytes / macrophages (myeloid cells). Considering other lineage differentiation (e.g., T cells, B cells, megakaryocytes, and erythroid cells), these data support the notion that miCD34 may be involved in the differentiation of HSCs into monocytes / macrophages (myeloid cells). +demonstrated multilineage differentiation of HSCs.

[0092] Example 11 miCD34 after treatment with platelet-derived mitochondria + The Notch signaling pathway contributes to HSC differentiation Notch signaling is well established as an essential regulator of HSC generation and differentiation. Specifically, the Notch signaling pathway plays a key role in T cell development and maturation at various stages. Both ex vivo and in vivo data support the role of miCD34 + To dissect the molecular mechanisms underlying mitochondrial processing, we investigated the effects of miCD34 on HSC differentiation, as shown in Figures 64-67. + We investigated the effect of Notch signaling during the differentiation of PB-IPCs into HSCs. Flow cytometry revealed that mitochondria expressed the Notch ligands Jagged1 (JAG1) (25.13% ± 16.0%), Jagged2 (JAG2) (68.04% ± 14.6%), and Delta-like 3 (DLL3) (69.3% ± 25.96%), but not DLL1 (2.21% ± 1.74%) or DLL4 (0.23% ± 0.09%) (Figure 64). After treatment with platelet-derived mitochondria, the expression levels of Notch receptors 1–4 in PB-IPCs were significantly upregulated (Figure 65). miCD34 in mitochondria-induced PB-IPCs + To investigate the role of Notch signaling in HSC differentiation, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine-t-butyl ester (DAPT) treatment was used to block γ-secretase, an enzyme required for releasing the Notch intracellular domain (NICD) into the nucleus to initiate gene transcription (Figure 66). In the mitochondria and DAPT (26.2% ± 5.68%) treated groups, CD34 + In contrast, DAPT treatment alone significantly increased the percentage of CD34 cells (Figure 67). + The ability to induce cells was very low (2.59% ± 0.13%), which is due to the presence of miCD34 +This indicates that mitochondria are required for HSC cell differentiation.

[0093] Having thus described the invention, it is asserted that what is new and desirably secured by Letters Patent is as follows: Another aspect of the present invention may be as follows. [1] A method for generating pluripotent cells, the method comprising: providing a sample of adult peripheral blood; isolating peripheral blood insulin-producing cells (PB-IPCs) from the adult peripheral blood sample by applying the adult peripheral blood sample to a hydrophobic surface and allowing the PB-IPCs to adhere to the hydrophobic surface; isolating mitochondria from the adult peripheral blood sample; treating the PB-IPCs with the mitochondria; the mitochondria enter the PB-IPC; The method, wherein the PB-IPCs have the invaded mitochondria configured to form pluripotent cells, corresponding to reprogramming of the PB-IPCs for pluripotent differentiation. 〔2〕 further comprising isolating peripheral blood-derived mononuclear cells (PBMCs) from the adult peripheral blood sample; The method of claim 1, wherein isolating PB-IPCs from the adult peripheral blood sample comprises applying the PBMCs to the hydrophobic surface and allowing the PB-IPCs to adhere to the hydrophobic surface. [3] The method according to [1], wherein isolating mitochondria from the adult peripheral blood comprises isolating platelets from the adult peripheral blood and isolating the mitochondria from the platelets. [4] The method according to [1], wherein isolating mitochondria from the adult peripheral blood comprises isolating PBMCs from the adult peripheral blood and isolating the mitochondria from the PBMCs. [5] The method according to [1], wherein isolating mitochondria from the adult peripheral blood comprises isolating plasma from the adult peripheral blood and isolating the mitochondria from the plasma. [6] The sample of adult peripheral blood comprises a first sample of adult peripheral blood from a first source and a second sample of adult peripheral blood from a second source; the first sample of adult peripheral blood is used to isolate the PB-IPCs; The method of claim 1, wherein a second sample of adult peripheral blood is used to isolate the mitochondria. [7] The method described in [1], further comprising a step in which the invaded mitochondria invade the nucleus of the PB-IPC. [8] The mitochondria contain an SDF-1 protein ligand; the PB-IPC nucleus comprises a nuclear membrane such as a CXCR4 protein receptor, The method of claim 7, wherein the invaded mitochondria that enter the nucleus of the PB-IPC contain the SDF-1 protein ligand that interacts with the CXCR4 protein receptor. [9] The method further comprises treating the PB-IPCs having the invaded mitochondria with a promoter for a desired differentiated cell; The method of claim 1, wherein the PB-IPCs having the invaded mitochondria develop into the desired differentiated cells.

[10] The method according to [9], wherein the desired differentiated cells are selected from the group consisting of macrophage cells, nerve cells, RPE cells, granulocyte cells, T cells, B cells, erythrocytes, megakaryocyte cells, platelet cells, bone marrow cells, stromal cells, osteoblasts, keratinocytes, hair follicle cells, glandular cells, endothelial cells, corneal endothelial cells, cardiac muscle cells, muscle cells, epithelial cells, hepatocytes, kidney cells, and pancreatic islet beta cells.

[11] A method for generating hematopoietic stem cell (HSC)-like cells, the method comprising: providing a sample of adult peripheral blood; isolating PB-IPCs from the adult peripheral blood sample by applying the adult peripheral blood sample to a hydrophobic surface and allowing the PB-IPCs to adhere to the hydrophobic surface; isolating mitochondria from the adult peripheral blood sample; treating the PB-IPCs with the mitochondria; the mitochondria enter the PB-IPC; the invaded mitochondria upregulate the HSC marker CD34 in the PB-IPCs; The method, wherein the PB-IPCs have the imported mitochondria configured to form HSC-like cells that are compatible with reprogramming of the PB-IPCs for hematopoietic differentiation. 〔12〕 further comprising isolating PBMCs from the adult peripheral blood sample; The method of claim 11, wherein isolating the PB-IPCs from the adult peripheral blood sample comprises applying the PBMCs to a hydrophobic surface and allowing the PB-IPCs to adhere to the hydrophobic surface.

[13] The method according to

[11] , wherein isolating mitochondria from the adult peripheral blood comprises isolating platelets from the adult peripheral blood and isolating the mitochondria from the platelets.

[14] The method according to

[11] , wherein isolating mitochondria from the adult peripheral blood comprises isolating PBMCs from the adult peripheral blood and isolating the mitochondria from the PBMCs.

[15] The method according to

[11] , wherein isolating mitochondria from the adult peripheral blood comprises isolating plasma from the adult peripheral blood and isolating the mitochondria from the plasma.

[16] The sample of adult peripheral blood comprises a first sample of adult peripheral blood from a first source and a second sample of adult peripheral blood from a second source; the first sample of adult peripheral blood is used to isolate the PB-IPCs; The method of claim 11, wherein a second sample of adult peripheral blood is used to isolate the mitochondria.

[17] The method further comprises treating the PB-IPCs having the invaded mitochondria with a hemocyte promoter; The method of claim 11, wherein the PB-IPCs having the invaded mitochondria develop into differentiated blood cells corresponding to the blood cell promoter.

[18] The method according to

[17] , wherein the differentiated blood cells are selected from the group consisting of macrophage cells, granulocyte cells, T cells, B cells, erythrocytes, megakaryocytes, and platelet cells.

[19] A method for generating pluripotent cells and for medical treatment, the method comprising: providing a sample of adult peripheral blood; isolating PBMCs from the adult peripheral blood sample; isolating PB-IPCs from the PBMCs by applying the PBMCs to a hydrophobic surface and allowing the PB-IPCs to adhere to the hydrophobic surface; isolating mitochondria from the adult peripheral blood sample; treating the PB-IPCs with the mitochondria, the mitochondria enter the PB-IPC; the PB-IPCs having the invaded mitochondria configured to form pluripotent cells for reprogramming of the PB-IPCs for pluripotent differentiation; treating the PB-IPCs having the invaded mitochondria with a promoter for a desired differentiated cell, developing the PB-IPCs with the invaded mitochondria into the desired differentiated cells; treating a patient with the desired differentiated cells.

[20] The method according to

[19] , wherein the desired differentiated cells are selected from the group consisting of macrophage cells, nerve cells, RPE cells, granulocyte cells, T cells, B cells, erythrocytes, megakaryocyte cells, platelet cells, bone marrow cells, stromal cells, osteoblasts, keratinocytes, hair follicle cells, glandular cells, endothelial cells, corneal endothelial cells, cardiac muscle cells, muscle cells, epithelial cells, hepatocytes, kidney cells, and pancreatic islet beta cells.

Claims

1. 1. A method for generating pluripotent cells, said method comprising: providing a sample of adult peripheral blood; isolating peripheral blood insulin-producing cells (PB-IPCs) from the adult peripheral blood sample by applying the adult peripheral blood sample to a hydrophobic surface and allowing the PB-IPCs to adhere to the hydrophobic surface; isolating mitochondria from the adult peripheral blood sample; treating the PB-IPCs with the mitochondria; The method, wherein the mitochondria enter the PB-IPCs, thereby reprogramming the PB-IPCs for pluripotent differentiation to form pluripotent cells.

2. further comprising isolating peripheral blood-derived mononuclear cells (PBMCs) from the adult peripheral blood sample; 2. The method of claim 1, wherein the step of isolating PB-IPCs from the adult peripheral blood sample comprises applying the PBMCs to the hydrophobic surface and allowing the PB-IPCs to adhere to the hydrophobic surface.

3. 2. The method of claim 1, wherein the step of isolating mitochondria from the adult peripheral blood sample comprises isolating platelets from the adult peripheral blood and isolating the mitochondria from the platelets.

4. 2. The method of claim 1, wherein the step of isolating mitochondria from the adult peripheral blood sample comprises isolating PBMCs from the adult peripheral blood and isolating the mitochondria from the PBMCs.

5. 2. The method of claim 1, wherein the step of isolating mitochondria from the adult peripheral blood sample comprises isolating plasma from the adult peripheral blood and isolating the mitochondria from the plasma.

6. the adult peripheral blood samples comprising a first sample of adult peripheral blood from a first source and a second sample of adult peripheral blood from a second source; the first sample of adult peripheral blood is used for the step of isolating the PB-IPCs; 10. The method of claim 1, wherein a second sample of adult peripheral blood is used for the step of isolating the mitochondria.

7. The method of claim 1, further comprising a step in which the invaded mitochondria enter the nucleus of the PB-IPC.

8. the mitochondria contain an SDF-1 protein ligand; the PB-IPC nucleus comprises a nuclear membrane containing the CXCR4 protein receptor; 8. The method of claim 7, wherein the invaded mitochondria that enter the nucleus of the PB-IPC contain the SDF-1 protein ligand that interacts with the CXCR4 protein receptor.

9. treating the PB-IPCs having the invaded mitochondria with a promoter for a desired differentiated cell; and The method of claim 1, further comprising the step of developing the PB-IPCs having the invaded mitochondria into the desired differentiated cells.

10. 10. The method of claim 9, wherein the desired differentiated cells are selected from the group consisting of macrophage cells, neuronal cells, RPE cells, granulocyte cells, T cells, B cells, erythrocytes, megakaryocyte cells, platelet cells, bone marrow cells, stromal cells, osteoblasts, keratinocytes, hair follicle cells, glandular cells, endothelial cells, corneal endothelial cells, cardiac myocytes, muscle cells, epithelial cells, hepatocytes, kidney cells, and pancreatic islet beta cells.

11. 1. A method for generating hematopoietic stem cell (HSC)-like cells, said method comprising: providing a sample of adult peripheral blood; isolating peripheral blood insulin-producing cells (PB-IPCs) from the adult peripheral blood sample by applying the adult peripheral blood sample to a hydrophobic surface and allowing the PB-IPCs to adhere to the hydrophobic surface; isolating mitochondria from the adult peripheral blood sample; treating the PB-IPCs with the mitochondria; the mitochondria enter the PB-IPC; The method, wherein the invaded mitochondria upregulate the HSC marker CD34 in the PB-IPCs, thereby reprogramming the PB-IPCs for hematopoietic differentiation to form HSC-like cells.

12. further comprising isolating PBMCs from the adult peripheral blood sample; 12. The method of claim 11, wherein the step of isolating the PB-IPCs from the adult peripheral blood sample comprises applying the PBMCs to the hydrophobic surface and allowing the PB-IPCs to adhere to the hydrophobic surface.

13. 12. The method of claim 11, wherein the step of isolating mitochondria from the adult peripheral blood sample comprises isolating platelets from the adult peripheral blood and isolating the mitochondria from the platelets.

14. 12. The method of claim 11, wherein the step of isolating mitochondria from the adult peripheral blood sample comprises isolating PBMCs from the adult peripheral blood and isolating the mitochondria from the PBMCs.

15. 12. The method of claim 11, wherein the step of isolating mitochondria from the adult peripheral blood sample comprises isolating plasma from the adult peripheral blood and isolating the mitochondria from the plasma.

16. the adult peripheral blood samples comprising a first sample of adult peripheral blood from a first source and a second sample of adult peripheral blood from a second source; the first sample of adult peripheral blood is used for the step of isolating the PB-IPCs; 12. The method of claim 11, wherein the second sample of adult peripheral blood is used for the step of isolating the mitochondria.

17. treating the PB-IPCs having the invaded mitochondria with a hemocyte promoter; and The method of claim 11, further comprising the step of developing the PB-IPCs having the invaded mitochondria into differentiated blood cells corresponding to the blood cell promoter.

18. 18. The method of claim 17, wherein the differentiated blood cells are selected from the group consisting of macrophage cells, granulocyte cells, T cells, B cells, erythrocytes, megakaryocyte cells, and platelet cells.

Images