(19)
(11)EP 3 177 303 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
29.07.2020 Bulletin 2020/31

(21)Application number: 15830332.1

(22)Date of filing:  07.08.2015
(51)Int. Cl.: 
A61K 35/12  (2015.01)
C12N 5/0735  (2010.01)
C12N 5/0775  (2010.01)
C12N 5/0789  (2010.01)
C12N 5/074  (2010.01)
(86)International application number:
PCT/US2015/044308
(87)International publication number:
WO 2016/022972 (11.02.2016 Gazette  2016/06)

(54)

APOPTOTIC BODIES

APOPTOTISCHE KÖRPER

CORPS APOPTOTIQUES


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 08.08.2014 US 201462035246 P

(43)Date of publication of application:
14.06.2017 Bulletin 2017/24

(73)Proprietors:
  • Alfred E. Mann Institute for Biomedical Engineering at the University of Southern California
    Los Angeles, CA 90089 (US)
  • University of Southern California
    Los Angeles, CA 90015 (US)

(72)Inventors:
  • SHI, Songtao
    Thousand Oaks, CA 91362 (US)
  • LIU, Dawei
    Philadelphia, PA 19104 (US)
  • QU, Cunye
    Charlottetown, PE C1A 3X5 (CA)

(74)Representative: Müller-Boré & Partner Patentanwälte PartG mbB 
Friedenheimer Brücke 21
80639 München
80639 München (DE)


(56)References cited: : 
US-A1- 2001 041 342
US-A1- 2006 127 383
US-A1- 2013 330 300
US-A1- 2005 202 098
US-A1- 2011 262 392
US-B1- 7 123 968
  
  • PEI-HSUN SUNG ET AL: "Apoptotic adipose-derived mesenchymal stem cell therapy protects against lung and kidney injury in sepsis syndrome caused by cecal ligation puncture in rats", STEM CELL RESEARCH & THERAPY, BIOMED CENTRAL LTD, LONDON, UK, vol. 4, no. 6, 25 December 2013 (2013-12-25), page 155, XP021175294, ISSN: 1757-6512, DOI: 10.1186/SCRT385
  • CHIA-LO CHANG ET AL: "Impact of apoptotic adipose-derived mesenchymal stem cells on attenuating organ damage and reducing mortality in Rat sepsis syndrome induced by cecal puncture and ligation", JOURNAL OF TRANSLATIONAL MEDICINE, BIOMED CENTRAL, vol. 10, no. 1, 7 December 2012 (2012-12-07), page 244, XP021134208, ISSN: 1479-5876, DOI: 10.1186/1479-5876-10-244
  • THUM THOMAS ET AL: "The Dying Stem Cell Hypothesis Immune Modulation as a Novel Mechanism for Progenitor Cell Therapy in Cardiac Muscle", JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY, vol. 46, no. 10, 2005, pages 1799-1802, XP029656539, ISSN: 0735-1097, DOI: 10.1016/J.JACC.2005.07.053
  • SAAS PHILIPPE ET AL: "Prospects of apoptotic cell-based therapies for transplantation and inflammatory diseases", IMMUNOTHERAPY, vol. 5, no. 10, October 2013 (2013-10), pages 1055-1073, XP009501748,
  
Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


Description


[0001] The present invention relates to compositions comprising an apoptotic body for use in a method of treating a tumor formation, wherein the tumor is hepatocellular carcinoma, multiple myeloma, or a combination thereof, and wherein the apoptotic body comprises an apoptotic stem cell.

[0002] This disclosure generally relates to cell therapies and cell therapy products. Some examples of cell therapy products include stem cell derived products and stem cells, such as those from mesenchymal, hematopoietic, embryonic, and umbilical cord blood; cancer therapies and immunotherapies, such as dendritic cell vaccines; activated T or B lymphocytes, monocytes, and modified or unmodified cancer cells, allogeneic pancreatic islet cells, bone-forming cells for bone regeneration, chondrocytes for cartilage repair, keratinocytes, fibroblasts, and hepatocytes. See, for example, Wonnacott K. "Cellular Therapy Products" Food and Drug Administration, Web Seminar Series, UCM273197.

[0003] An example of a cell therapy product is a composition comprising mesenchymal stem cells (MSCs). The MSCs possess multipotent differentiation potential and capability to regulate immune response. For example, see Uccelli et al. "Mesenchymal stem cells in health and disease" (2008) Nat. Rev. Immunol. 8:726-736; Nauta et al. "Immunomodulatory properties of mesenchymal stromal cells" (2007) Blood 110:3499-3506; and Aggarwal et al. "Human mesenchymal stem cells modulate allogeneic immune cell responses" (2005) Blood 105:1815-1822. Cell types that MSCs have been shown to differentiate into in vitro or in vivo include osteoblasts, chondrocytes, myocytes, adipocytes, endotheliums, and beta-pancreatic islets supporting cells.

[0004] MSCs have a large capacity for self-renewal while maintaining their multipotency. The standard test to confirm multipotency is differentiation of the cells into osteoblasts, adipocytes, and chondrocytes and possibly neuron-like cells. However, the degree to which the culture will differentiate varies among individuals and how differentiation is induced, e.g. chemical vs. mechanical. The capacity of cells to proliferate and differentiate is known to decrease with the age of the donor, as well as the time in culture.

[0005] It was disclosed that human MSCs may avoid allorecognition, interfere with dendritic cell and T-cell function and generate a local immunosuppressive microenvironment by secreting cytokines. It was also disclosed that the immunomodulatory function of human MSCs may be enhanced when the cells are exposed to an inflammatory environment characterized by the presence of elevated local interferon-gamma levels.

[0006] Thus, MSCs may be used in tissue regeneration and immune therapies. For example, see Le Blanc et al. "Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study" (2008) Lancet 371:1579-1586; Sun et al. "Mesenchymal stem cell transplantation reverses multiorgan dysfunction in systemic lupus erythematosus mice and humans" (2009) Stem Cells 27:1421-1432; and Akiyama et al. "Mesenchymal-stem-cell-induced immunoregulation involves FAS-ligand-/FAS-mediated T cell apoptosis" (2012) Cell Stem Cell 10:544-555.

[0007] In this context, Sung et al. (Sung, P.-H. et al.; Stem Cell Research & Therapy, 4(155); 2013; pp. 1-20) describes an apoptotic adipose-derived mesenchymal stem cell therapy against lung and kidney injury in sepsis syndrome in a rat animal model. Further, Chang et al. (Chang, C.-L. et al.; Journal of Translational Medicine, 10(244); 2012; pp. 1-14) describes the impact of apoptotic adipose-derived mesenchymal stem cells on attenuating organ damage and reducing mortality in sepsis syndrome in a rat animal model.

[0008] However, there still exists a need for other sources of cell therapy products and new approaches for preparation thereof, and use of such products for medical treatments.

[0009] The present invention relates to the following items:
  1. 1. A composition comprising an apoptotic body for use in a method of treating a tumor formation, wherein the tumor is hepatocellular carcinoma, multiple myeloma, or a combination thereof, and wherein the apoptotic body comprises an apoptotic stem cell.
  2. 2. The composition for use of item 1, wherein the apoptotic stem cell comprises an apoptotic mesenchymal stem cell, an apoptotic embryonic stem cell, an apoptotic fetal stem cell, an apoptotic adult stem cell, an apoptotic amniotic stem cell, an apoptotic cord blood stem cell, an apoptotic induced pluripotent stem cell, an apoptotic bone marrow-derived mesenchymal stem cell, an apoptotic dental pulp stem cell, an apoptotic stem cell from human exfoliated deciduous teeth, an apoptotic periodontal ligament stem cell, an apoptotic dental follicle stem cell, an apoptotic tooth germ progenitor cell, an apoptotic stem cell from the apical papilla, an apoptotic oral epithelial progenitor/stem cell, an apoptotic gingiva-derived mesenchymal stem cell, an apoptotic periosteum-derived stem cell, an apoptotic salivary gland-derived stem cell, an apoptotic body derived from cultured mesenchymal stem cell, an apoptotic body derived from an uncultured gingiva-derived mesenchymal stem cell, an apoptotic dental pulp stem cell, an apoptotic bone-marrow-derived stem cell, or a combination thereof.
  3. 3. The composition for use of item 1, wherein the apoptotic stem cell is obtained by incubating a stem cell in a serum-free medium for a time period in the range of 1 hour to 1,000 hours.
  4. 4. The composition for use of item 1, wherein the apoptotic stem cell is obtained by heating a stem cell at a temperature in the range of 30° C to 100°C for a predetermined period of time.
  5. 5. The composition for use of item 1, wherein the apoptotic stem cell is obtained by treating a stem cell with staurosporine in an amount in the range of 1 nM staurosporine to 10,000 nM staurosporine for a time period in the range 1 hour to 1,000 hours.
  6. 6. The composition for use of item 1, wherein the apoptotic stem cell is obtained by treating a stem cell in a serum free medium with staurosporine in an amount in the range of 1 nM staurosporine to 10,000 nM staurosporine for a time period in the range of 1 hour to 1,000 hours.
  7. 7. The composition for use of item 1, wherein the apoptotic stem cell is obtained by irradiating a stem cell at a wavelength in the range of 100 nm to 400 nm for a time period in the range of 0.1 minute to 1,000 minutes.


[0010] This disclosure relates to apoptotic bodies. The disclosure particularly relates to a composition comprising the apoptotic bodies. The disclosure further relates to preparation of apoptotic bodies from stem cells. Preferably, the apoptotic bodies are prepared from multipotent or pluripotent stem cells, and not from totipotent stem cells. More preferably, the apoptotic bodies are prepared from multipotent stem cells, and not from pluripotent or totipotent stem cells. The disclosure also relates to medical treatments comprising the use of the composition comprising the apoptotic bodies.

[0011] This disclosure relates to a composition. This composition may be suitable for a treatment of a mammal. This composition may comprise an apoptotic body. The apoptotic body may comprise a apoptotic stem cell.

[0012] In this disclosure, the apoptotic stem cell may comprise any apoptotic stem cell. For example, the apoptotic stem cell may comprise an apoptotic mesenchymal stem cell, an apoptotic embryonic stem cell, an apoptotic fetal stem cell, an apoptotic adult stem cell, an apoptotic amniotic stem cell, an apoptotic cord blood stem cell, an apoptotic induced pluripotent stem cell, or a combination thereof. For example, the apoptotic stem cell may comprise an apoptotic mesenchymal stem cell. For example, the apoptotic stem cell may comprise an apoptotic bone marrow-derived mesenchymal stem cell, an apoptotic dental pulp stem cell, an apoptotic stem cell from human exfoliated deciduous teeth, an apoptotic periodontal ligament stem cell, an apoptotic dental follicle stem cell, an apoptotic tooth germ progenitor cell, an apoptotic stem cell from the apical papilla, an apoptotic oral epithelial progenitor/stem cell, an apoptotic gingiva-derived mesenchymal stem cell, an apoptotic periosteum-derived stem cell, an apoptotic salivary gland-derived stem cell, or a combination thereof. For example, the apoptotic stem cell may comprise an apoptotic body derived from cultured mesenchymal stem cell, an apoptotic body derived from an uncultured gingiva-derived mesenchymal stem cell, an apoptotic dental pulp stem cell, an apoptotic bone-marrow-derived stem cell, or a combination thereof.

[0013] In this disclosure, the apoptotic stem cell may be obtained by the apoptosis of a stem cell, For example, the apoptosis of a stem cell may be induced by a starvation method, an ultra-violet irradiation method, a thermal stress method, a staurosporine method, or a combination thereof.

[0014] For example, the apoptotic stem cell may be obtained by incubating a stem cell in a serum-free medium for a time period in the range of 1 hour to 1,000 hours. Or, the apoptotic stem cell may be obtained by incubating a stem cell in a serum-free medium for a time period in the range of 10 hours to 100 hours.

[0015] In another example, the apoptotic stem cell may be obtained by heating a stem cell at a temperature in the range of 30° C to 100°C for a predetermined period of time. Or, the apoptotic stem cell may be obtained by heating a stem cell at a temperature in the range of 30° C to 100°C for a time period in the range of 1 minute to 1,000 minutes. Or, the apoptotic stem cell may be obtained by heating a stem cell at a temperature in the range of 30° C to 100°C for a time period in the range of 10 minutes to 100 minutes. Or, the apoptotic stem cell may be obtained by heating a stem cell at a temperature in the range of 40° C to 70°C for a predetermined time. Or, the apoptotic stem cell may be obtained by heating a stem cell at a temperature in the range of 40° C to 70°C for a time period in the range of 1 minute to 1,000 minutes. Or, the apoptotic stem cell may be obtained by heating a stem cell at a temperature in the range of 40° C to 70°C for a time period in the range of 10 minute to 100 minutes.

[0016] Yet, in another example, the apoptotic stem cell may be obtained by treating a stem cell with staurosporine in an amount in the range of 1 nm staurosporine to 10,000 nM staurosporine for a time period in the range 1 hour to 1,000 hours. Or, the apoptotic stem cell may be obtained by treating a stem cell with staurosporine in an amount in the range of 1 nm staurosporine to 10,000 nM staurosporine for a time period in the range 5 hours to 100 hours. Or, the apoptotic stem cell may be obtained by treating a stem cell with staurosporine in an amount in the range of 100 nm staurosporine to 1,000 nM staurosporine for a time period in the range 1 hour to 1,000 hours. Or, the apoptotic stem cell may be obtained by treating a stem cell with staurosporine in an amount in the range of 100 nm staurosporine to 1,000 nM staurosporine for a time period in the range 5 hours to 100 hours. Or, the apoptotic stem cell may be obtained by treating a stem cell in a serum free medium with staurosporine in an amount in the range of 1 nm staurosporine to 10,000 nM staurosporine for a time period in the range of 1 hour to 1,000 hours. Or, the apoptotic stem cell may be obtained by treating a stem cell in a serum free medium with staurosporine in an amount in the range of 1 nm staurosporine to 10,000 nM staurosporine for a time period in the range of 5 hours to 100 hours. Or, the apoptotic stem cell may be obtained by treating a stem cell in a serum free medium with staurosporine in an amount in the range of 100 nm staurosporine to 1,000 nM staurosporine for a time period in the range of 1 hour to 1,000 hours. Or, the apoptotic stem cell may be obtained by treating a stem cell in a serum free medium with staurosporine in an amount in the range of 100 nm staurosporine to 1,000 nM staurosporine for a time period in the range of 5 hours to 100 hours.

[0017] Yet, in another example, the apoptotic stem cell may be obtained by irradiating a stem cell at a wavelength in the range of 100 nm to 400 nm for a time period in the range of 0.1 minute to 1,000 minutes. Or, the apoptotic stem cell may be obtained by irradiating a stem cell at a wavelength in range of 100 nm to 400 nm for a UV lamp for a time period in the range of 1 minute to 100 minutes.

[0018] This disclosure also relates ta a method for preparation of the composition of this disclosure. This preparation method may, for example, comprise inducing apoptosis of a stem cell and thereby preparing the apoptotic body comprising the apoptotic stem cell.

[0019] In this preparation method, the stem cell may comprise any stem cell. For example, the stem cell may comprise an embryonic stem cell, a fetal stem cell, an adult stem cell, an amniotic stem cell, a cord blood stem cell, an induced pluripotent stem cell, or a combination thereof. For example, the stem cell may comprise a mesenchymal stem cell. For example, the stem cell may comprise a bone marrow-derived mesenchymal cell, a dental pulp stem cell, a stem cell from human exfoliated deciduous teeth, a periodontal ligament stem cell, a dental follicle stem cell, a tooth germ progenitor cell, a stem cell from the apical papilla, an oral epithelial progenitor/stem cell, a gingiva-derived mesenchymal stem cell, a periosteum-derived stem cell, a salivary gland-derived stem cell, or a combination thereof. For example, the stem cell may comprise a cultured mesenchymal stem cell, an uncultured gingiva-derived mesenchymal stem cell, a dental pulp stem cell, a bone-marrow-derived stem cell, or a combination thereof.

[0020] For example, in this disclosure, the apoptosis may be induced by a starvation method, an ultra-violet irradiation method, a thermal stress method, a staurosporine method, or a combination thereof.

[0021] In this disclosure, the apoptotic body may, for example, be collected by centrifugation.

[0022] The preparation method may further comprise identifying, quantifying, and/or isolating the apoptotic body by a fluorescent microscopy technique, a flow cytometry technique, a centrifugation technique, or a combination thereof.

[0023] This disclosure also relates to a method of treating a human subject. This treatment method may, for example, comprise administrating an effective amount of a composition, comprising an apoptotic body comprising an apoptotic stem cell of a human, into the human subject and thereby treating the human subject.

[0024] In this disclosure, the treating may comprise treating a disease. The disease may, for example, may comprise an inflammatory, an autoimmune disease, bone loss caused by ovariectomy, a tumor formation and/or a wound.

[0025] For example, the inflammatory and/or autoimmune disease may be graft-versus host disease (GvHD), diabetes, rheumatoid arthritis (RA), autoimmune encephalomyelitis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), periodontitis, inflammatory bowel disease (IBD), mucositis, colitis, sepsis, the like, or a combination thereof. For example, the inflammatory and/or autoimmune disease may comprise colitis. For example, the inflammatory and/or autoimmune disease may comprise systemic lupus erythematosus. For example, the disease may be bone loss caused by ovariectomy. For example, the disease may be a tumor formation. For example, the tumor may be hepatocellular carcinoma, multiple myeloma, or a combination thereof. For example, the disease may be a wound.

[0026] In the treatment method, the apoptotic stem cell of a human may, for example, comprise an apoptotic mesenchymal stem cell of a human, an apoptotic embryonic stem cell of a human, an apoptotic fetal stem cell of a human, an apoptotic adult stem cell of a human, an apoptotic amniotic stem cell of a human, an apoptotic cord blood stem cell of a human, an apoptotic induced pluripotent stem cell of a human, or a combination thereof. For example, the apoptotic stem cell of a human may comprise an apoptotic mesenchymal stem cell of a human. For example, the apoptotic stem cell of a human may comprise an apoptotic bone marrow-derived mesenchymal cell of a human, an apoptotic dental pulp stem cell of a human, an apoptotic stem cell from human exfoliated deciduous teeth of a human, an apoptotic periodontal ligament stem cell of a human, an apoptotic dental follicle stem cell of a human, an apoptotic tooth germ progenitor cell of a human, an apoptotic stem cell from the apical papilla of a human, an apoptotic oral epithelial progenitor/stem cell of a human, an apoptotic gingiva-derived mesenchymal stem cell of a human, an apoptotic periosteum-derived stem cell of a human, an apoptotic salivary gland-derived stem cell of a human, or a combination thereof. For example, the apoptotic stem cell of a human may comprise an apoptotic body derived from cultured mesenchymal stem cell of a human, an apoptotic body derived from an uncultured gingiva-derived mesenchymal stem cell of a human, an apoptotic dental pulp stem cell of a human, an apoptotic bone-marrow-derived stem cell of a human, or a combination thereof.

[0027] This disclosure also relates to a method of improvement of stem cell properties ("improvement method"). The improvement method may comprise using an effective amount of a composition of this disclosure. The improvement method may comprise an apoptotic body comprising an apoptotic stem cell. The improvement method may improve: (a) colony formation of bone marrow mesenchymal stem cells in vivo and/or in vitro; (b) immunomodulation of bone marrow mesenchymal stem cells in vivo and/or in vitro; (c) osteogenesis of bone marrow mesenchymal stem cells in vivo and/or in vitro; (d) adipogenesis of bone marrow mesenchymal stem cells in vivo and/or in vitro; or a combination thereof. For example, the improvement method may comprise improving the colony formation of bone marrow mesenchymal stem cells in vivo and/or in vitro. For example, the improvement method may comprise improving the immunomodulation of bone marrow mesenchymal stem cells in vivo and/or in vitro. For example, the improvement method may comprise improving the osteogenesis of bone marrow mesenchymal stem cells in vivo and/or in vitro. For example, the improvement method may comprise improving the adipogenesis of bone marrow mesenchymal stem cells in vivo and/or in vitro.

[0028] Any combination of products such as compositions comprising apoptotic bodies, methods of their preparation, and methods of their use that are described herein may also be made and followed.

[0029] These, as well as other components, steps, features, objects, benefits, and advantages, will now become clear from a review of the following detailed description of illustrative embodiments, the accompanying drawings, and the claims.

[0030] The drawings disclose illustrative embodiments. They do not set forth all embodiments. Other embodiments may be used in addition or instead. Details which may be apparent or unnecessary may be omitted to save space or for more effective illustration. Conversely, some embodiments may be practiced without all of the details which are disclosed. When the same numeral appears in different drawings, it refers to the same or like components or steps.

[0031] The figures show:

FIG. 1. Schematic demonstration of GMSCs. The cell apoptosis was induced by the staurosporine method. The cell apoptosis was detected by using the light microscope and the flow cytometric analysis.

FIG. 2. Schematic demonstration of apoptotic body collection.

FIG. 3. Apoptotic body identification by fluorescent microscopy.

FIG. 4. Schematic demonstration of the relative counting method and an experimental result of an apoptotic body counting obtained by using this method.

FIGs. 5A-B. Apoptotic body purification and sorting using the flow cytometric sorting method. DRAQ5 was used as fluorescent marker.

FIG. 6. Schematic demonstration of apoptotic body purification and microscopy of the cells purified by this method.

FIGs. 7A-B. Schematic demonstration of systemic infusion of a mouse by apoptotic bodies and harvesting of BMMSCs. Experimental CFU numbers are shown in this figure.

FIGs. 8A-B. BMMSC proliferation in vitro by apoptotic body treatment. (A) Cell microscopy and (B) cell percentage.

FIGs. 9A-B. Osteogenic differentiation potential of the BMMSCs treated with apoptotic bodies in vitro.(A) Alizarin staining and (B) Immunoblot analysis.

FIGs. 10A-B. Mineralized tissue formation potential of the BMMSCs treated with the apoptotic bodies. (A) Tissue microscopy and (B) Bone volume area.

FIGs. 11A-B. Adipogenesis potential of the BMMSCs treated with the apoptotic bodies.(A) Oil-red O staining and (B) Immunoblot analysis.

FIGs. 12A-B. Improvement of immunomodulation potential of BMMSCs in vitro by apoptotic body treatment.

FIGs. 13A-D. Treatment of the colitis mice with apoptotic bodies.

FIGs. 14A-I. Treatment of the ovariectomized mice with apoptotic bodies.

FIGs. 15A-G. Treatment of the systemic lupus erythematosus (SLE) mice with apoptotic bodies.

FIGs. 16A-B. Apoptotic body infusion inhibited solid tumor growth in mouse model of hepatocellular carcinoma. (A) Xenograft tumors with apoptotic body treatment showed reduced tumor volume. (B) HE staining showed apoptotic body-treated tumor induced infiltration of hematopoietic cells compared to control tumor.

FIGs. 17A-B. Apoptotic body treatment reduced in vitro tumor cell survival. (A) Cell survival analysis indicated co-culture with apoptotic bodies reduced the tumor cell survival in a dosage-dependent manner. (B) Microarray analysis of global gene expression pattern indicated apoptotic body treatment increased the cell apoptosis and lysosome function.

FIG. 18. Apoptotic body infusion improved the survived rate of mouse models of multiple myeloma. Kaplan-Meier survival analysis of the mouse models indicated apoptotic body treatment significant improved the mouse life span.

FIGs. 19A-E. Apoptotic body (AB) enteral infusion improves DSS-induced colitis. (A) Since DSS administration can induce colitis in mice, we infused about 1x107 AB or bone marrow mesenchymal stem cells (MSCs) at about 3 days post DSS induction and harvest samples at about 10 days post DDS induction to evaluate therapeutic effect of AB and use MSC enteral infusion as a positive control. The apoptotic body treatment showed: (B) significant therapeutic effect than MSC group in rescuing body weight and (C) reduction of histological activity index in the colon and reduction. (D and E) Compared with the colitis group, AB treatment showed a significant effect in reducing the levels of Th1 and Th17 cells in the mice at about 10 days post-colitis induction. These experimental results demonstrate that AB enteral infusion can be used as a novel approach in the treatment of immune disorder such as colitis.

FIGs. 20A-C. AB infusion improves wound healing in mouse ear punch model. (A) about 5x106 AB was infused into mice wither before or after the ear punch. (B and C) Ear hole area of AB-treated group was significantly reduced when compared to un-treated control group at about 7 days post ear punch.



[0032] Illustrative embodiments are now discussed. Other embodiments may be used in addition or instead. Certain details which may be apparent to a person of ordinary skill in the art or unnecessary may have been omitted from this disclosure to to provide for a clear and concise disclosure. Conversely, some embodiments may be practiced without all of the details which are disclosed.

[0033] The following acronyms and abbreviations were used in this disclosure.

AB: Apoptotic Bodies

BMMSC: Bone-Marrow-derived Mesenchymal Stem Cell

BMD: Bone Mineral Density

CFU-F: Colony Forming Unit - Fibroblast

DFSC: Dental Follicle Stem Cell

DPSC: Dental Pulp Stem Cell

DSS: Dextran Sulfate Sodium

FACS: Fluorescence Assisted Cell Sorting

GMSC: Gingiva-derived Mesenchymal Stem Cell

GvHD: Graft-versus-Host Disease

HE: Hematoxylin and Eosin

HA/TCP: Hydroxyapatite Tricalcium Phosphate

MSC: Mesenchymal Stem Cell

nM: nano-Molar

OESC: Oral Epithelial Progenitor/Stem Cell

PDLSC: Periodontal Ligament Stem Cell

PSC: Periosteum-derived Stem cell

SCAP: Stem Cells from the Apical Papilla

SGSC: Salivary Gland-derived Stem Cell

SHED: Stem Cell from Human Exfoliated Deciduous Teeth

SLE: Systemic Lupus Erythematosus

TGPC: Tooth Germ Progenitor Cell

UV: Ultra-Violet

µm: micrometer



[0034] This disclosure relates to apoptotic bodies. The disclosure particularly relates to a composition comprising the apoptotic bodies. The disclosure further relates to preparation of apoptotic bodies from stem cells. The disclosure also relates to medical treatments comprising using the composition comprising the apoptotic bodies.

[0035] Apoptotic bodies may be vesicles that are approximately 0.5-10 µm in diameter and generated by cells undergoing programmed cell death, termed apoptosis, which may initiate with cell shrinkage and condensation of the nuclear chromatin, followed by membrane blebbing. Cellular organelles, condensed chromatin and their fragmentations may be encapsulated into the plasma membrane to form apoptotic bodies. Apoptotic bodies may be collected from apoptotic cells by series centrifuge, as disclosed in Example 3. Apoptotic bodies may be characterized as phosphatidylserine exposure on the surface membrane that may be detected with Annexin V staining and other surface marker, such as TSP-1. About 10% apoptotic bodies may contain genetic material that may be labeled with Hoechst 33342.

[0036] This disclosure also relates to an apoptotic body therapy (or apoptotic body treatment). The apoptotic body treatment may include, but not limited to, diagnosis, treatment, cure, healing, mitigation, or prevention of a disease or injury in, and/or cosmetic treatment of a mammal. The mammal may be a human. The mammal may be a non-human animal, such as a non-human primate, a horse, a sheep, a cattle, a hog, a dog, a cat, and a goat.

[0037] This disclosure further relates to methods of using the composition comprising apoptotic bodies in the treatment of tumors, and inflammatory and/or autoimmune diseases ("treatment methods"). Examples of the tumors may be hepatocellular carcinoma and multiple myeloma. Examples of such diseases are graft-versus-host disease (GvHD), diabetes, rheumatoid arthritis (RA), autoimmune encephalomyelitis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), periodontitis, inflammatory bowel disease (IBD), alimentary tract mucositis induced by chemo- or radiotherapy, colitis, and sepsis. Another example of the treatment method may be the treatment of the bone loss caused by ovariectomy.

[0038] This disclosure also relates to of using the composition comprising apoptotic bodies in the improvement of BMMSCs ("improvement methods"). Examples of such uses to improve BMMSCs include use for the improvement of colony formation of bone marrow mesenchymal stem cells in vivo or in vitro, improvement of osteogenesis of bone marrow mesenchymal stem cells in vivo or in vitro, improvement of adipogenesis of bone marrow mesenchymal stem cells in vivo or in vitro, improvement of immunomodulation of bone marrow mesenchymal stem cells in vivo or in vitro, and combinations thereof.

[0039] The composition useful for a treatment may comprise an apoptotic body. The composition may comprise and apoptotic body that may be prepared by apoptosis of a stem cell. In one example, the apoptotic body may comprise an apoptotic stem cell. The apoptotic stem cell may be a stem cell prepared by apoptosis a stem cell. In all embodiments of this disclosure, it is preferred that the apoptotic bodies are prepared from multipotent or pluripotent stem cells, and not from totipotent stem cells. More preferably, the apoptotic bodies are prepared from multipotent stem cells, and not from pluripotent or totipotent stem cells.

[0040] In this disclosure, the stem cell may be a stem cell of a mammal. The mammal may be a human. The mammal may be a non-human animal, such as a non-human primate, a horse, a sheep, a cattle, a hog, a dog, a cat, and a goat.

[0041] In this disclosure, the stem cell of a human may be used to prepare an apoptotic stem cell. Such human derived apoptotic stem cells may be used in treatment of human subjects.

[0042] This disclosure relates to a composition. The composition may comprise an apoptotic body. The composition may comprise a cell culture comprising an apoptotic body. The composition may be a drug or a biologic formulation. The composition may be used in the apoptotic body treatment.

[0043] In this disclosure, the composition may comprise an apoptotic body comprising apoptotic stem cells.

[0044] Stem cells, which are suitable for preparation of the exemplary composition, may be any stem cell of any mammal. For example, the stem cells may be stem cells of a mammal that undergoes the treatment (i.e. apoptotic body treatment comprising using apoptotic bodies comprising apoptotic autologous stem cells, "autologous apoptotic body treatment"). Or, the stem cells may be stem cells of a mammal other than the mammal that undergoes the treatment (i.e. allogeneic body treatment comprising using apoptotic bodies comprising apoptotic allogeneic stem cells, "allogeneic apoptotic body treatment").

[0045] Following are examples of the stem cells that are suitable in preparation of the apoptotic bodies comprising apoptotic stem cells.

[0046] The stem cell may be any stem cell. Examples of stem cells may be embryonic stem cells, fetal stem cells, adult stem cells, amniotic stem cells, cord blood stem cells, induced pluripotent stem cells, or combinations thereof.

[0047] An example of stem cells may comprise mesenchymal stem cells (MSCs). Examples of mesenchymal stem cells may comprise bone marrow-derived mesenchymal cells (BMMSCs), dental pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSCs), dental follicle stem cells (DFSCs), tooth germ progenitor cells (TGPCs), stem cells from the apical papilla (SCAPs), oral epithelial progenitor/stem cells (OESCs), gingiva-derived mesenchymal stem cells (GMSCs), periosteum-derived stem cells (PSCs), salivary gland-derived stem cells (SGSCs), and combinations thereof.

[0048] For in detail disclosure of such stem cells, tissues suitable for their harvest, methods of their isolation from such tissues, their expansion, and methods of administration of such stem cells for a treatment, see, for example, Egusa et al. "Stem Cells in Dentistry - Part I: Stem Cell Sources" J. Prosthodontic Res. (212) v56, p151-165; Shi et al. "A composition of stem cells having highly expressed Fas ligand," WO2015038665A1; Atsuta et al. "Mesenchymal stem cells inhibit multiple myeloma cells via the Fas/Fas ligand pathway" Stem Cell Research & Therapy, 2013, 4:111; Le et al. "Gingiva Derived Stem Cell and Its Application in Immunomodulation and Reconstruction", U.S. 2012/0128636A1 ; Shi et al. "A Composition of Mesenchymal Stem Cells", WO2014210037; Shi et al. "Compositions and Treatment Methods for Mesenchymal Stem Cell-Induced Immunoregulation," US20150104428 A1; Shi et al." High Telomerase Activity Bone Marrow Mesenchymal Stem Cells, Methods of Producing the Same and Pharmaceuticals and Treatment Methods Based Thereon," US20130330300 A1; and Shi et al. "Methods and Compositions for Improved Tissue Regeneration by Suppression of Interferon-Gamma and Tumor Necrosis Factor-Alpha," US20140154220 A1.

[0049] In another example, cultured and/or uncultured MSCs may be used in preparation of apoptotic bodies. In another example, uncultured and/or GMSCs, DPSCs or a combination thereof may be used. Yet, in another example, cultured and/or uncultured BMMSCs may be used. Still, in another example, a combination of these uncultured and/or cultured stem cells may be used.

[0050] The apoptotic stem cells may thereby be prepared from the stem cells. Thus, examples of such apoptotic stem cells may be an apoptotic mesenchymal stem cell, an apoptotic embryonic stem cell, an apoptotic fetal stem cell, an apoptotic adult stem cell, an apoptotic amniotic stem cell, an apoptotic cord blood stem cell, an apoptotic induced pluripotent stem cell, and a combination thereof. For example, the apoptotic stem cells may be a apoptotic bone marrow-derived mesenchymal cell, an apoptotic dental pulp stem cell, an apoptotic stem cell from human exfoliated deciduous teeth, an apoptotic periodontal ligament stem cell, an apoptotic dental follicle stem cell, an apoptotic tooth germ progenitor cell, an apoptotic stem cell from the apical papilla, an apoptotic oral epithelial progenitor/stem cell, an apoptotic gingiva-derived mesenchymal stem cell, an apoptotic periosteum-derived stem cell, an apoptotic salivary gland-derived stem cell, or a combination thereof. In other examples, the apoptotic stem cell may comprise an apoptotic body derived from cultured mesenchymal stem cell, an apoptotic body derived from an uncultured gingiva-derived mesenchymal stem cell, an apoptotic dental pulp stem cell, an apoptotic bone-marrow-derived stem cell, or a combination thereof.

[0051] Apoptosis of the stem cells may be achieved by using variety of methods. For example, the apoptotic stem cell may be prepared by inducing apoptosis of the stem cell. The examples of the stem cells apoptosis method may include a stem cell starvation method, a ultra-violet irradiation method, a thermal stress method, a staurosporine method, and combinations thereof.

[0052] The cell apoptosis may be detected by using variety of methods. Examples of the detection methods may be the detection by using a light microscope, the detection by using flow cytometry, and combinations thereof.

[0053] The apoptotic bodies may be collected by centrifugation.

[0054] The apoptotic bodies may be identified, quantified, and and/or purified by a fluorescent microscopy technique, a flow cytometry technique, and/or a centrifugation technique.

[0055] This disclosure also relates to a method of preparation ("preparation method"). The preparation method may, for example, comprise obtaining (or harvesting) a tissue comprising stem cells, preparing an apoptotic body using the tissue comprising stem cells, and preparing a composition comprising an apoptotic body. In another example, the preparation method may comprise obtaining (or harvesting) a tissue comprising stem cells, separating the tissue into cells, sorting stem cells from the cells, preparing an apoptotic body using the sorted stem cells, and preparing a composition comprising the apoptotic body. Yet, in another example, the preparation method may comprise obtaining (or harvesting) a tissue comprising stem cells, separating the tissue into cells, sorting stem cells from the cells, expanding the stem cells, preparing an apoptotic body using the expanded stem cells, and preparing a composition comprising the apoptotic body. The preparation method may further comprise culturing the separated cells before sorting the stem cells. The preparation method may further comprise isolating a stem cell.

[0056] This disclosure also relates to a method of administration of the composition comprising apoptotic bodies ("administration method"). The administration method may comprise any method suitable for administration of a cell or a tissue. The composition comprising the apoptotic bodies suitable for the treatment purposes may be administered (or delivered) in various ways. For example, they may be infused, injected at various sites, or surgically implanted..

[0057] Suitable compositions include, for example, an apoptotic body (or cell) suspension in a liquid (preferably aqueous) medium, and apoptotic bodies in aggregated form with or without solid supports or encapsulating materials. The liquid medium may be a pharmaceutically acceptable liquid medium. The liquid medium may be suitable for injection. Suitable liquid media are generally well known and include, for example, normal saline (with or without glucose and/or potassium), Ringer's solution, Lactated Ringer's solution, and Hartmann's solution.

[0058] The composition may be administered in an amount effective to treat a mammal. The apoptotic body amount (or dose) of the composition may be an amount effective to treat a mammal. The amount and/or concentration of apoptotic bodies in the composition can be selected to provide for convenient administration of an amount (of the composition that is acceptable for the mammal being treated. For example, a liquid composition that contain a high concentration of apoptotic bodies is suitable for injection of a relatively small volume into solid tissue, while a liquid composition that contains a lower concentration of apoptotic bodies may be advantageous for administration by intravenous infusion. Suitable liquid compositions can contain, for example, about 1×101 apoptotic bodies/mL to about 1×1010 apoptotic bodies/mL, or about 1×105 apoptotic bodies/mL to about 1×107 apoptotic bodies/mL, or about 1×106 apoptotic bodies/mL to 5×106 apoptotic bodies/mL. Aggregated or solid compositions can contain, for example, about 1×101 apoptotic bodies/mg to about 1×1010 apoptotic bodies/g.

[0059] The composition may further comprise a carrier. The carrier may be suitable to host the apoptotic body. Example of the carrier may be in the form of matrices, tissues, fibers, beads, or other materials.

[0060] For therapeutic purposes, an effective amount of the composition may be administered to a mammal in need thereof. An "effective amount" in an amount that produces the desired effect under the conditions of administration, for example, an amount sufficient to inhibit inflammation, inhibit an immune or autoimmune response, inhibit bone loss, inhibit tumor growth or metastisis, promote wound healing, or promote colony formation, osteogenesis or adipogenesis by BMMSCs.

[0061] Generally, the effective amount may include about 1×101 apoptotic bodies to about 1×1010 apoptotic bodies, or about 1×105 apoptotic bodies to about 1×107 apoptotic bodies, or about 1×106 apoptotic bodies to 5×106 apoptotic bodies per administration. The exact dosage of apoptotic cells to be administered may dependent upon a variety of factors, including the age, weight, and sex of the mammal, the disease being treated, and the extent and severity thereof. A clinician of ordinary skill can determine the appropriate dose and method for administration based on these and other considerations.

[0062] In this disclosure, the apoptotic stem cell may be obtained by the apoptosis of a stem cell induced by a starvation method, an ultra-violet irradiation method, a thermal stress method, a staurosporine method, or a combination thereof. These methods may be carried out in a serum free medium.

[0063] For example, to induce cell apoptosis by starvation method, the apoptotic stem cell may be obtained by incubating stem cell in a serum-free medium for a time period in the range of 1 hour to 1,000 hours, or in the range of 10 hours to 100 hours.

[0064] For example, to induce cell apoptosis by a thermal stress method, the apoptotic stem cell may be obtained by heating a stem cell at a predetermined temperature for a predetermined period of time. For example, to induce cell apoptosis by a thermal stress method, the apoptotic stem cell may be obtained by heating a stem cell at a temperature in the range of 30° C to 100°C, or in the range of 40° C to 70°C; for a period of time in the range of 1 minute to 1,000 minutes, or 10 minutes to 100 minutes. The thermal stress method may be carried out in a serum free medium.

[0065] For example, to induce cell apoptosis by a staurosporine method, the apoptotic stem cell may be obtained by treating a stem cell with a predetermined amount of staurosporine for a predetermined period of time. For example, the apoptotic stem cell may be obtained by treating a stem cell with staurosporine in an amount in the range of 1 nm staurosporine to 10,000 nM staurosporine, or in an amount in the range of 1 nm staurosporine to 10,000 nM staurosporine; for a time period in the range 1 hour to 1,000 hours, or in the range 5 hours to 100 hours. The staurosporine method may be carried out in a serum free medium.

[0066] For example, to induce cell apoptosis by a ultra-violet (UV) method, a stem cell may be irradiated at a predetermined wavelength for a predetermined period of time. For example, the stem cell may be irradiated at a wavelength in the range of 100 nm to 400 nm for a time period in the range of 0.1 minute to 1,000 minutes, or in the range of 1 minute to 100 minutes. The UV radiation method may be carried out in a serum free medium.

[0067] Any combination of products such as compositions comprising apoptotic bodies, compositions comprising apoptotic bodies comprising apoptotic stem cells, methods of their preparation, and methods of their use that are described herein may also be made and followed.

[0068] Other examples of this disclosure are as follows.

EXAMPLE 1. PROCEDURE TO INDUCE CELL APOPTOSIS.



[0069] The cell apoptosis may be achieved by using variety of methods.

[0070] For example, in a starvation method, cells substantially comprising confluent MSCs were incubated in serum-free medium for about 72 hours to induce cell apoptosis. In this method, the cells may comprise close to 100% confluent MSCs.

[0071] In a UV irradiation method, i.e. another exemplary method, in, cultured MSCs may be treated with a UV lamp for a time period in the range of 5 minutes to 10 minutes to induce apoptosis The UV lamp may be operated at about 110 V, about 50 Hz, and about 180 W.

[0072] In a thermal stress method, i.e. another exemplary method, cultured MSCs may be incubated at a temperature varying in the range of 45° C to 50°C for a time period in the range of 40 minutes to 60 minutes to induce apoptosis.

[0073] In a staurosporine method, i.e. another exemplary method, cells substantially comprising confluent MSCs were treated with about 250 nM staurosporine in the serum-free medium for about 15 hours to induce cell apoptosis. In this method, the cells may comprise close to 100% confluent MSCs.

[0074] Above methods may be combined to induce cell apoptosis. For example, the starvation method may be combined with the UV irradiation method, and/or the thermal stress to induce apoptosis.

EXAMPLE 2. CELL APOPTOSIS DETECTION.



[0075] The cell apoptosis may be detected by using variety of methods. For example, the cell shrinkage and pyknosis may be detected by using a light microscope. Also, Annexin V and 7-AAD double positive apoptotic cells may be detected using flow cytometric analysis.

[0076] In one example, the cell apoptosis was induced by staurosporine. The cell apoptosis was detected by using the light microscope and the flow cytometric analysis. The results are shown in FIG. 1. Cell unattached from culture dish and shrinkage could be observed under light microscope. Compared with the control group, after staurosporine treatment, 7AAD+ or Annexin V+ or double positive apoptosis cell populations increased from 3.3% to 96.8%.

EXAMPLE 3. APOPTOTIC BODY COLLECTION.



[0077] Culture medium from induced apoptotic cells was collected in a sterile centrifuge tube (about 15 ml in about 50 ml tube). First, the dead cells and the cell debris were removed by centrifugation at about 800 g for about 10 minutes. Then, the supernatant was transferred into about 1.5 ml EP tubes. The supernatant was further centrifuged at about 16, 000 g for about 30 minutes to obtain the apoptotic bodies as a pellet. To eliminate the reagent or serum effect in the medium, the pellet was re-suspended and washed by PBS and re-centrifuged at about 16, 000 g for about 30 minutes. This example is schematically shown in FIG. 2.

EXAMPLE 4. APOPTOTIC BODY IDENTIFICATION.



[0078] In this example, the apoptotic bodies were identified by using a fluorescent microscopy technique. The results are shown in FIG. 3. The membrane of apoptotic bodies were labeled with PKH26 and the genetic material was labeled with Hoechst 33342 as described in the manufacturer's instructions. The diameter of apoptotic bodies varied in the range of 0.5 µm to 10 µm, with the average size about 3.014±1.1093 µm. Less than 10% of apoptotic bodies contained genetic materials. Surface markers Annexin V, 7-AAD and TSP of apoptotic bodies may be identified by flow cytometric analysis.

EXAMPLE 5. APOPTOTIC BODY QUANTITATION AND PURIFICATION.



[0079] A relative counting method was used for apoptotic body quantitation. First, a known amount of fluorescent dye labeled beads ("counting beads") were added into apoptotic body suspension. Then, the relative ratio of apoptotic bodies and counting beads were detected by using the flow cytometric analysis. This method is schematically demonstrated in FIG. 4. The number of apoptotic bodies were calculated based on the known amount of the counting beads according to the following formula:



[0080] The harvest rate of apoptotic bodies to the cells varied in the range of 5 to 10.

EXAMPLE 6. APOPTOTIC BODY PURIFICATION BY FLOW CYTOMETRIC PURIFICATION.



[0081] In this example, the apoptotic bodies were further purified by using the flow cytometric sorting technique. These purified apoptotic bodies may be used in preparation of the composition of this disclosure, wherein the apoptotic body may be the purified apoptotic body.

[0082] Genetic material in apoptotic bodies was labeled by DRAQ5 as the marker for the flow cytometric sorting. Apoptotic bodies were separated into two populations. Results are shown in FIGs. 5A-B. The DRAQ5 positive population contained apoptotic bodies with the genetic material and the DRAQ5 negative population contained apoptotic bodies with no genetic material. 3.8% apoptotic bodies with genetic material can be sorted out, meanwhile, 43.13% apoptotic bodies without genetic material can be collected during flow cytometric sorting.

EXAMPLE 7. APOPTOTIC BODY PURIFICATION BY DIFFERENTIAL CENTRIFUGATION.



[0083] In this example, the apoptotic bodies were further purified by using the differential centrifugation technique. This method of purification of apoptotic bodies is schematically shown in FIG. 6. These purified apoptotic bodies may be used in preparation of the composition of this disclosure, wherein the apoptotic body may be the purified apoptotic body.

[0084] Based on the different sedimentation rate, large sized apoptotic bodies were separated using a low centrifugal force. First, the pelleted apoptotic bodies were re-suspended with PBS and then centrifuged at about 4,000 g for about 5 minutes. The diameters of pelleted apoptotic bodies varied in the range of 4 µm to 10 µm, and more than 80% of apoptotic bodies contained genetic material. The results are shown in FIG. 6.

EXAMPLE 8. APOPTOTIC BODIES IMPROVE BMMSC COLONY FORMING NUMBERS IN VIVO.



[0085] C3H mice were subjected to systemic infusion with apoptotic bodies at day 0 and day 2. The dose of each systemic infusion was 5×106 apoptotic body cells. BMMSCs were harvested from the mice at day 7. The mice infused with apoptotic bodies generated higher numbers of BMMSC CFU-F than wild type (wt) mice did, as shown by toluidine blue staining. The CFU-F numbers of apoptotic bodies treatment group increased from 42 colonies/ 1 million cells to 57 colonies / 1 million cells. See FIGs. 7A-B. These experimental results demonstrated that apoptotic bodies can improve BMMSC colony forming numbers in vivo.

EXAMPLE 9. APOPTOTIC BODIES IMPROVE BMMSC COLONY FORMING NUMBERS IN VITRO.



[0086] In this example, BMMSCs were treated with apoptotic bodies and analyzed by incorporation of a BrdU assay. It was found that the number of BrdU-positive (BrdU+) cells significantly increased in BMMSCs treated with the apoptotic bodies when compared with that of untreated BMMSCs. See FIGs. 8A-B. These experimental results demonstrated that apoptotic bodies can improve BMMSC colony forming numbers in vitro.

EXAMPLE 10. APOPTOTIC BODIES IMPROVE OSTEOGENESIS OF BMMSCs IN VITRO.



[0087] In this example, the BMMSCs were treated with apoptotic bodies in vitro. The osteogenic differentiation potential of BMMSCs were assessed at six weeks after the osteogenic induction by Alizarin red staining. The Alizarin-red-positive (Alizarin Red+) area, in terms of mineralization nodes, was calculated as a percentage of total area. Immunoblot analysis revealed that expression levels of OCN had the same trend. The experimental results are shown in FIGs. 9A-B.

[0088] The BMMSCs treated with the high-dose of apoptotic bodies formed significantly increased amounts of mineralized nodules as compared with those of the untreated BMMSCs and the BMMSCs treated with the low-dose of apoptotic bodies. These experimental results demonstrated that the osteogenic differentiation potential of BMMSCs can be improved by apoptotic body treatment in vitro. These experimental results further demonstrated that this improvement potential can be increased by increasing the dose of the apoptotic bodies.

EXAMPLE 11. APOPTOTIC BODIES IMPROVE OSTEOGENESIS OF BMMSCs IN VIVO.



[0089] In this example, the BMMSCs treated with the apoptotic bodies in vitro were transplanted subcutaneously into immunocompromised mice by using hydroxyapatite tricalcium phosphate (HA/TCP) as a carrier. Experimental results shown in FIGs. 10A-B indicated that the BMMSCs treated with the apoptotic bodies formed more mineralized tissue when compared with the untreated BMMSCs. These experimental results demonstrated that the mineralized tissue formation potential of BMMSCs can be improved by their in vivo treatment with the apoptotic bodies.

EXAMPLE 12. APOPTOTIC BODIES IMPROVE ADIPOGENESIS OF BMMSCs IN VITRO.



[0090] In this example, the BMMSCs were treated with apoptotic bodies in vitro. The adipogenesis potential of the BMMSCs were assessed at four weeks after the adipogenesis induction by Oil-red O staining. Numbers of Oil-red O-positive (Oil-Red-O+) cells were calculated as a percentage of total cells. The experimental results shown in FIGs. 11A-B indicated that the BMMSCs treated with the apoptotic bodies had an elevated potential to differentiate into adipocytes. Immunoblot assay indicated that the expression levels of adipocyte-specific molecules LPL and PPARy2 had the same trend. These experimental results demonstrated that the adipogenesis potential of the BMMSCs can be improved by their in vitro treatment with the apoptotic bodies.

EXAMPLE 13. APOPTOTIC BODIES IMPROVE IMMUNOMODULATION OF BMMSCs IN VITRO.



[0091] BMMSC can directly induce active T cells apoptosis as the immunomodulation properties. BMMSC/T-cell co-culture system showed that apoptotic bodies treated BMMSCs had significantly increased capacity to induce AnnexinV+7AAD+ double positive apoptotic T cells, when compared to the untreated BMMSCs. Experimental results are shown in FIGs. 12A-B.

EXAMPLE 14. APOPTOTIC BODIES HAVE THERAPEUTIC EFFECT ON INDUCTIVE COLITIS MICE.



[0092] In this example, first group of mice with DSS-induced experimental colitis were infused with a dose of 5 x 106 apoptotic bodies ("AB infusion"). For the positive control, a second group of mice with DSS-induced experimental colitis were infused with a dose of 5 x 105 BMMSCs ("MSC infusion"). A third group of mice with DSS-induced experimental colitis were left untreated ("colitis"). The first and the second mice groups were infused three days after the induction of the colitis. Weights loss of three mice groups were recorded over a period of 10 days, starting from the induction of the colitis. Results are shown in FIGs. 13A-D.

[0093] The apoptotic body treatment exerted more significant restoration of disease phenotype, body weight loss, in the colon and reduction of histological activity index compared with those of the colitis group. Compared with the colitis group, the apoptotic body treatment showed a significant effect in reducing the levels of Th17 cells in the mice at 10 days post-colitis induction. These experimental results demonstrated that the apoptotic bodies can be used in the treatment of immune disorder diseases like colitis.

EXAMPLE 15. APOPTOTIC BODIES HAVE THERAPEUTIC EFFECT ON OVARIECTOMIZED MICE.



[0094] In this example, a first group of ovariectomized mice were infused with a dose of 5 x 106 apoptotic bodies ("OVX+AB"). For the positive control, a second group of ovariectomized mice were infused with a dose of 5 x 105 BMMSCs ("OVX+MSC"). A third group of ovariectomized mice were left untreated ("OVX"). A fourth group of non- ovariectomized mice were also left untreated ("Sham"). The first and the second mice groups were infused two weeks after the induction of the ovariectomy. Results are shown in FIGs. 14A-I.

[0095] Hematoxylin and eosin (HE) staining showed that both the BMMSC and the apoptotic body treatment significantly rescued the bone loss in ovariectomized mice. The apoptotic body treatment significantly improved bone mineral density (BMD) in femurs BV/TV compared to that of the untreated ovariectomized mice. Also, the apoptotic body infusion rescued CFU-F numbers, population doublings, osteogenesis capability and adipogenesis capability of OVX-derived bone marrow MSCs. Meanwhile, the apoptotic body infusion reduced the level of Th1 and Th17 t cells.

EXAMPLE 16. APOPTOTIC BODIES HAVE THERAPEUTIC EFFECT ON SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) MICE.



[0096] In this example, MRL/lpr mice was used as a systemic lupus erythematosus model. A first group of MRL/lpr mice were infused with apoptotic bodies ("Lpr+AB"). For the positive control, a second group of MRL/lpr mice were infused with BMMSCs ("Lpr+MSC"). A third group of MRL/lpr mice were left untreated ("Ipr"). A fourth group of C3H mice were also left untreated ("C3H"). Results are shown in FIGs. 15A-G.

[0097] The apoptotic body infusion rescued mesenchymal stem cell (MSC) functional deficiency and reconstructed trabecular bone structure of MRL/lpr mice. The apoptotic body infusion significantly improved bone mineral density (BMD) in femurs BV/TV compared to that of the untreated MRL/lpr mice. The apoptotic body infusion reduced levels of autoantibodies and improved renal function in MRL/lpr mice. Levels of C3, anti-dsDNA in peripheral blood decreased in the apoptotic body infused group compared with those of the untreated group, while albumin level was rescued. The apoptotic body infusion reduced Th17 T cell level and recovered Treg/Th17 ratio.

EXAMPLE 17. APOPTOTIC BODIES HAVE THERAPEUTIC EFFECT ON TUMOR.



[0098] The therapeutic effect of apoptotic bodies was evaluated in both solid tumors and hematopoietic tumor. For this evaluation, the mouse tumor models of hepatocellular carcinoma and multiple myeloma were established.

[0099] For mouse model of hepatocellular carcinoma, xenografts of hepatocellular carcinoma were generated by intramuscular injection of HepG2 cells. Briefly, the mouse model was conducted in weight-matched, about 8-week-old female NOD/SCID mice (Jackson Lab, Bar Harbor, ME, USA). HepG2 hepatocellular carcinoma cells (ATCC, Manassas, VA, USA) were cultured in Dulbecco's Modified Eagle Medium (Invitrogen Co., Carlsbad, CA, USA) supplemented by about 10% fetal bovine serum (Summit Biotechnology, Fort Collins, CO, USA) and antibiotics (Invitrogen Co., Carlsbad, CA, USA) at about 37 °C, about 5% CO2. HepG2 cells (about 1 × 106 cells/10 grams body weight) suspended in PBS were injected intramuscularly in the hind legs of NOD/SCID mice and PBS injection served as negative control.

[0100] To test the effect of apoptotic bodies on in situ carcinoma, diethylnitrosamine as a carcinogen was injected in B6C3F1 mice for induction of in situ hepatocelluar carcinoma. Briefly, about 4 week old B6C3F1 male mice were used to generate the carcinoma model. A single dosage of diethylnitrosamine (about 100 mg/gram body weight) was intraperitoneally administrated to the mice.

[0101] For multiple myeloma model, the 5TGM1 MM cell line, subcloned from a stroma-independent cell line from parent murine 5 T33 (IgG2bK) multiple myeloma, was grown in long-term suspension culture in Isocove's modified Dulbecco's medium (Invitrogen Co., Carlsbad, CA, USA) with about 10% fetal bovine serum and antibiotics. 5TGM1 multiple myeloma model was conducted in weight-matched about 10-week-old female NOD/SCID mice. The mice were housed in isolator cages, and autoclaved chow and acidified water were provided ad libitum. Disseminated multiple myeloma was induced by the intravenous inoculation of 5TGM1 cells (about 6 × 106/10 grams body weight) in about 200 µl PBS through the tail vein.

[0102] The apoptotic bodies were delivered in vivo as follows. Mouse with xenograft tumors of hepatocellular carcinoma and multiple myeloma were randomized to treated group and control group. After tumor cell inoculation for about 1 week, a single dose of apoptotic bodies (about 3X106 /10 grams body weight) were delivered via tail vein injection (treated group) and PBS injection served as control.

[0103] For mouse models of in situ hepatocellular carcinoma, apoptotic bodies (about 3 × 106/10 grams body weight) were monthly administrated into the mice via tail vein.

[0104] The therapeutic effects of the apoptotic bodies were assessed as follows. Xenograft tumors of hepatocellular carcinoma were surgically harvested about 4 weeks after tumor inoculation. The therapeutic effect of apoptotic bodies was assessed by measuring the volume of the tumors by the formula: volume=length × width2 × 0.52. To reveal the histology of the tumors with or without apoptotic body treatment, the tissue was fixed in about 4% paraformaldehyde for about 48 hours and subsequently embedded in paraffin for Hematoxyin-Eosin staining.

[0105] For assessment of the therapeutic effects of apoptotic bodies in multiple myeloma models, the 8-week survival rates of the mice in different groups were compared. The results were analyzed by Kaplan-Meier survival method.

[0106] In vivo cytotoxicity of the apoptotic bodies on tumor cells were determined as follows. HepG2 hepatocellular carcinoma cells were seeded in 24-well plate at the concentration of about 1 × 105 cells/well. About 12 hours after cell seeding, the cells were treated by apoptotic bodies (about 1 × 106 apoptotic bodies/well) or PBS and different concentration of apoptotic bodies was applied in the cell culture for evaluation of the cytotoxicity. The cell survival was assayed by an automated cell counter (Bio-rad, Hercules, CA, USA) about 2 days after the treatment of apoptotic bodies and dead cells were excluded by staining of trypan blue (Invitrogen Co., Carlsbad, CA, USA).

[0107] Results of the apoptotic treatment of tumors were as follows. Intramuscular inoculation of HepG2 cells successfully generated tumor growth in NOD/SCID mice. To test the therapeutic effect in the tumor model, apoptotic bodies were systemically infused into the mice with xenograft tumor. The results of tumor volume measurement showed that tumors in the mice that received apoptotic body treatment were significantly smaller than that in the mice without the treatment (FIG. 16A). Xenograft tumors generated by HepG2 cells were filled by progressive tumor cell growth and after apoptotic body treatment, the tumors were infiltrated by hematopoietic cells with large area of necrosis (FIG. 16B). To confirm the therapeutic effect, in vitro co-culture assay indicated obvious cytotoxicity of apoptotic bodies to HepG2 cells, which was in a dosage-dependent manner (FIG. 17A). Comparison of global gene expression pattern indicated after apoptotic body treatment, the tumor cells underwent enhanced function of apoptosis and lysosome (FIG. 17B).

[0108] In mouse model of multiple myeloma, the mice that received systemic apoptotic body infusion survived significantly longer than control mice, indicating the therapy effectively inhibit the disease progression (FIG. 18).

EXAMPLE 18. APOPTOTIC BODY ENTERAL INFUSION IMPROVES COLITIS.



[0109] Apoptotic body (AB) infusion improves DSS-induced colitis, as shown in FIG. 19. Since DSS infusion can induce colitis in mice, we infused about 1x107 AB or bone marrow mesenchymal stem cells (MSCs) enterally at about 3 days post DSS induction and harvest samples at about 10 days post DDS induction to evaluate therapeutic effect of AB and use MSC infusion as a positive control (FIG. 19A). The apoptotic body treatment showed significant therapeutic effect than MSC group in rescuing body weight (FIG. 19B). The apoptotic body treatment also showed reduction of histological activity index in the colon (FIG. 19C). Compared with the colitis group, AB treatment showed a significant effect in reducing the levels of Th1 and Th17 cells in the mice at about 10 days post-colitis induction (FIGs. 19D-E). These experimental results demonstrated that AB infusion can be used as a novel approach in the treatment of immune disorder such as colitis.

EXAMPLE 19. APOPTOTIC BODY INFUSION IMPROVES WOUND HEALING.



[0110] AB infusion improves wound healing in mouse ear punch model, as shown in FIGs. 20A-C. About 5x106 AB was infused into mice wither before or after the ear punch (FIG. 20A). Ear hole area of AB-treated group was significantly reduced when compared to un-treated control group at about 7 days post ear punch (FIG. 20B-C).

[0111] Any combination of products such as compositions comprising apoptotic bodies, methods of their preparation, and methods of their use that are described herein may also be made and followed.


Claims

1. A composition comprising an apoptotic body for use in a method of treating a tumor formation, wherein the tumor is hepatocellular carcinoma, multiple myeloma, or a combination thereof, and wherein the apoptotic body comprises an apoptotic stem cell.
 
2. The composition for use of claim 1, wherein the apoptotic stem cell comprises an apoptotic mesenchymal stem cell, an apoptotic embryonic stem cell, an apoptotic fetal stem cell, an apoptotic adult stem cell, an apoptotic amniotic stem cell, an apoptotic cord blood stem cell, an apoptotic induced pluripotent stem cell, an apoptotic bone marrow-derived mesenchymal stem cell, an apoptotic dental pulp stem cell, an apoptotic stem cell from human exfoliated deciduous teeth, an apoptotic periodontal ligament stem cell, an apoptotic dental follicle stem cell, an apoptotic tooth germ progenitor cell, an apoptotic stem cell from the apical papilla, an apoptotic oral epithelial progenitor/stem cell, an apoptotic gingiva-derived mesenchymal stem cell, an apoptotic periosteum-derived stem cell, an apoptotic salivary gland-derived stem cell, an apoptotic body derived from cultured mesenchymal stem cell, an apoptotic body derived from an uncultured gingiva-derived mesenchymal stem cell, an apoptotic dental pulp stem cell, an apoptotic bone-marrow-derived stem cell, or a combination thereof.
 
3. The composition for use of claim 1, wherein the apoptotic stem cell is obtained by incubating a stem cell in a serum-free medium for a time period in the range of 1 hour to 1,000 hours.
 
4. The composition for use of claim 1, wherein the apoptotic stem cell is obtained by heating a stem cell at a temperature in the range of 30° C to 100°C for a predetermined period of time.
 
5. The composition for use of claim 1, wherein the apoptotic stem cell is obtained by treating a stem cell with staurosporine in an amount in the range of 1 nM staurosporine to 10,000 nM staurosporine for a time period in the range 1 hour to 1,000 hours.
 
6. The composition for use of claim 1, wherein the apoptotic stem cell is obtained by treating a stem cell in a serum free medium with staurosporine in an amount in the range of 1 nM staurosporine to 10,000 nM staurosporine for a time period in the range of 1 hour to 1,000 hours.
 
7. The composition for use of claim 1, wherein the apoptotic stem cell is obtained by irradiating a stem cell at a wavelength in the range of 100 nm to 400 nm for a time period in the range of 0.1 minute to 1,000 minutes.
 


Ansprüche

1. Zusammensetzung, umfassend ein Apoptosekörperchen zur Verwendung in einem Verfahren zur Behandlung einer Tumorbildung, wobei der Tumor ein hepatozelluläres Karzinom, ein multiples Myelom oder eine Kombination davon ist, und wobei das Apoptosekörperchen eine apoptotische Stammzelle umfasst.
 
2. Zusammensetzung zur Verwendung nach Anspruch 1, wobei die apoptotische Stammzelle eine apoptotische mesenchymale Stammzelle, eine apoptotische embryonale Stammzelle, eine apoptotische fötale Stammzelle, eine apoptotische adulte Stammzelle, eine apoptotische amniotische Stammzelle, eine apoptotische Nabelschnurblut-Stammzelle, eine apoptotisch induzierte pluripotente Stammzelle, eine apoptotisch aus dem Knochenmark stammende mesenchymale Stammzelle, eine apoptotische Zahnmark-Stammzelle, eine apoptotische Stammzelle aus ausgefallenen menschlichen Milchzähnen, eine apoptotische Parodontalband-Stammzelle, eine apoptotische Dentalfollikel-Stammzelle, eine apoptotische Zahnkeim-Vorläuferzelle, eine apoptotische Stammzelle aus der apikalen Papille, eine apoptotische orale epitheliale Vorläufer-/Stammzelle, eine apoptotische mesenchymale Stammzelle aus dem Zahnfleisch, eine apoptotische Stammzelle aus dem Periost, eine apoptotische Stammzelle aus der Speicheldrüse, ein Apoptosekörperchen, das aus kultivierten mesenchymalen Stammzellen stammt, ein Apoptosekörperchen, das aus einer nicht kultivierten, aus dem Zahnfleisch stammenden mesenchymalen Stammzelle gewonnen wird, eine apoptotische Dentalpulpen-Stammzelle, eine apoptotische Knochenmark-Stammzelle oder eine Kombination davon umfasst.
 
3. Zusammensetzung zur Verwendung nach Anspruch 1, wobei die apoptotische Stammzelle durch Inkubieren einer Stammzelle in einem serumfreien Medium für eine Zeitdauer im Bereich von 1 Stunde bis 1000 Stunden erhalten wird.
 
4. Zusammensetzung zur Verwendung nach Anspruch 1, wobei die apoptotische Stammzelle durch Erhitzen einer Stammzelle bei einer Temperatur im Bereich von 30°C bis 100°C für eine vorbestimmte Zeitdauer erhalten wird.
 
5. Zusammensetzung nach Anspruch 1, wobei die apoptotische Stammzelle durch Behandlung einer Stammzelle mit Staurosporin in einer Menge im Bereich von 1 nM Staurosporin bis 10.000 nM Staurosporin über einen Zeitraum im Bereich von 1 Stunde bis 1.000 Stunden erhalten wird.
 
6. Zusammensetzung zur Verwendung nach Anspruch 1, wobei die apoptotische Stammzelle durch Behandlung einer Stammzelle in einem serumfreien Medium mit Staurosporin in einer Menge im Bereich von 1 nM Staurosporin bis 10.000 nM Staurosporin über einen Zeitraum im Bereich von 1 Stunde bis 1.000 Stunden erhalten wird.
 
7. Zusammensetzung zur Verwendung nach Anspruch 1, wobei die apoptotische Stammzelle durch Bestrahlung einer Stammzelle bei einer Wellenlänge im Bereich von 100 nm bis 400 nm für eine Zeitdauer im Bereich von 0,1 Minuten bis 1000 Minuten erhalten wird.
 


Revendications

1. Composition comprenant un corps apoptotique à utiliser dans un procédé de traitement d'une formation tumorale, où la tumeur est un carcinome hépatocellulaire, un myélome multiple, ou une combinaison de ceux-ci, et où le corps apoptotique comprend une cellule souche apoptotique.
 
2. Composition à utiliser selon la revendication 1, dans laquelle la cellule souche apoptotique comprend une cellule souche mésenchymateuse apoptotique, une cellule souche embryonnaire apoptotique, une cellule souche fÅ“tale apoptotique, une cellule souche adulte apoptotique, une cellule souche amniotique apoptotique, une cellule souche apoptotique de sang de cordon, une cellule souche pluripotente induite apoptotique, une cellule souche mésenchymateuse apoptotique dérivée de moelle osseuse, une cellule souche apoptotique de pulpe dentaire, une cellule souche apoptotique de dents de laits exfoliées humaines, une cellule souche apoptotique de ligament périodontique, une cellule souche apoptotique de follicule dentaire, une cellule souche apoptotique de germe dentaire, une cellule souche apoptotique de papille apicale, cellule souche/progénitrice épithéliale orale apoptotique, une cellule souche mésenchymateuse apoptotique dérivée de la gencive, une cellule souche apoptotique dérivée du périoste, une cellule souche apoptotique dérivée des glandes salivaires, un corps apoptotique dérivé de cellules souches mésenchymateuses en culture, un corps apoptotique dérivé de cellules souches mésenchymateuses de gencive non cultivées, une cellule souche apoptotique de pulpe dentaire, une cellule souche apoptotique dérivée de moelle osseuse ou une combinaison de ceux-ci.
 
3. Composition à utiliser selon la revendication 1, dans laquelle la cellule souche apoptotique est obtenue par incubation d'une cellule souche dans un milieu exempt de sérum pendant une période allant de 1 heure à 1000 heures.
 
4. Composition à utiliser selon la revendication 1, dans laquelle la cellule souche apoptotique est obtenue par chauffage d'une cellule souche à une température située dans la plage allant de 30°C à 100°C pendant une période prédéterminée.
 
5. Composition à utiliser selon la revendication 1, dans laquelle la cellule souche apoptotique est obtenue par traitement d'une cellule souche avec la staurosporine en une quantité située dans l'intervalle allant de 1 nM de staurosporine à 10 000 nM de staurosporine pendant une période allant de 1 heure à 1000 heures.
 
6. Composition à utiliser selon la revendication 1, dans laquelle la cellule souche apoptotique est obtenue par traitement d'une cellule souche dans un milieu exempt de sérum, avec la staurosporine en une quantité située dans l'intervalle allant de 1 nM de staurosporine à 10 000 nM de staurosporine pendant une période allant de 1 heure à 1000 heures.
 
7. Composition à utiliser selon la revendication 1, dans laquelle la cellule souche apoptotique est obtenue par irradiation d'une cellule souche à une longueur d'onde située dans la plage allant de 100 nm à 400 nm pendant une période allant de 0,1 minute à 1000 minutes.
 




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REFERENCES CITED IN THE DESCRIPTION



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