Patent Application
20170247660
1. Field of the Invention
The present invention relates to multipotent progenitor cells, a method for preparing the progenitor cells from uterine corpus, and uses of the progenitor cells.
2. Description of the Related Art
Progenitor cell therapy is likely the best answer to curing degenerative diseases such as Parkinson's disease and ischemic diseases such as stroke and myocardial infarction—all disease entities highly correlated with aging populations. Current sources of human progenitor cells (also known as stem cells) include pluripotent stem cells (PSCs) such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPS), as well as adult stem cells (ASCs) such as bone marrow mesenchymal stem cells (BMMSCs) and neural stem cells. However, each source is not without its drawbacks for clinical use: PSCs have ethical and tumorigenicity concerns (Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., and Jones, J. M. (1998). Embryonic stem cell lines derived from human blastocysts. Science 282, 1145-1147; Yamanaka, S. (2009). A fresh look at iPS cells. Cell 137, 13-17), while ASCs are very rare cells requiring invasive procedures for procurement with cell numbers and stem cell functions decreasing with age (Rao, M. S., and Mattson, M. P. (2001). Stem cells and aging: expanding the possibilities. Mech Ageing Dev 122, 713-734) and ex vivo expansion (Ho, P. J., Yen, M. L., Tang, B. C., Chen, C. T., and Yen, B. L. (2013). H2O2 accumulation mediates differentiation capacity alteration, but not proliferative decline, in senescent human fetal mesenchymal stem cells. Antioxid Redox Signal 18, 1895-1905) (“Ho et al”)).
Uterus is an organ in the female reproductive tract whose function is to carry the human fetus and nurture until term. Capable of intense growth to accommodate the physiological demands of pregnancy, the uterus corpus is mainly comprised of smooth muscle cells—the myometrium—and is highly vascularized. Removal of the uterus, or a hysterectomy, is the most common surgical procedure performed on non-pregnant woman; in the United States, one of three women past the age of 60 has had a hysterectomy (Schaffer, J. I., and Word, A. (2002). Hysterectomy—still a useful operation. N Engl J Med 347, 1360-1362; Carlson, K. J., Nichols, D. H., and Schiff, I. (1993). Indications for hysterectomy. N Engl J Med 328, 856-860; Frequently Asked Questions: Hysterectomy. (2009). Office on Women's Health, U.S. Department of Health & Human Services).
Ono et al. reported cells termed “myometrial side-population stem cells (myoSPs)” can be isolated from human uterus (Ono, M., Maruyama, T., Masuda, H., Kajitani, T., Nagashima, T., Arase, T., Ito, M., Ohta, K., Uchida, H., Asada, H., et al. (2007). Side population in human uterine myometrium displays phenotypic and functional characteristics of myometrial stem cells. Proc Natl Acad Sci USA 104, 18700-18705 (“Ono et al, PNAS 2007”)). Myometrial tissue has to be incubated in medium with minimal enzyme addition (0.02%) for 4-16 hours with subsequent filtration (2 times) and gradient selection (Ficollpaque), then further trypsinized. MyoSPs were characterized by the ability to efflux Hoechst 33342 dye, which show up on flow cytometric analysis as a “side population,” and also exibit CD31 (+), CD34(+), and CD44(−). However, MyoSPs are unable to differentiate into chondrogenic cells, nor reported to differentiate into neurogenic cells.
Galvez et al., In Vivo 2009, WO2010/057965 and WO2011/042547A1 disclose myometrial-derived mesenchymal stem cells and isolation method thereof. The isolated cells termed “adult myometrial precursors (AMPs)” which were isolated to from myometrial tissue. The myometrial tissue was incubated in serum-free medium and only fragments with small vessels were selected for further culturing. Therefore, the method is for selecting endothelial-like cells, and the results showing high CD31(+) percentage of these isolated AMPs (>99.7% for mouse AMPs and >90% for human AMPs) can be explained accordingly. The cell surface marker profile of AMPs are CD73(−), CD31(+) and HLA-DR (+). Only differentiation capacity of mouse AMPs were disclosed and the mouse AMPs possess osteogenesis, adipogenesis and neurogenesis, but no chondrogenesis was reported.
Therefore, it still needs an alternative source for obtaining progenitor cells and a method for preparing the progenitor cells.
The present application describes a method for preparing progenitor cells comprising obtaining a tissue sample containing myometrium from uterus, treating the tissue sample with an enzyme to remove fibrous tissue, and culturing the treated tissue sample to obtain the progenitor cells, wherein the progenitor cell is multipotent and immunomodulatory.
The present application also provides a progenitor cells obtained from the above method.
The present application further provides a method for treating a degenerative disease, an ischemic disease or a disease caused by abnormal immune response comprising administering progenitor cells prepared according to the above method to a patient subjecting the disease.
The present application describes a method for preparing progenitor cells comprising obtaining a tissue sample containing myometrium from uterus. The isolated progenitor cell is multipotent and immunomodulatory.
As used herein, the term “myometrium” refers to tissue derived from the middle layer of the uterine wall.
As used herein, the term “uterus” encompasses the cervical canal and uterine cavity.
The multipotent and immunomodulatory progenitor cells of the present application can be obtained from any tissue sample containing myometrium of any suitable source from any suitable animal. In one embodiment, the suitable animal is an mammal such as a rodent, primate, carnivora, artiodactyla and the like, preferably a primate.
In one embodiment, the tissue sample can be obtained from non-pathological post-natal uterus.
In one preferred embodiment, the tissue sample is obtained from the human uterine corpus.
In one embodiment, the uterus can be from hysterectomies. Hysterectomy is the most common surgical procedure performed on non-pregnant woman. The method of the present application allows for the efficient isolation of uterine myometrium-derived multipotent progenitors (MDMPs) from post-hysterectomy specimens—which is to be discarded—with multilineage differentiation capacity, immunomodulation, and high proliferative potential. In addition, the method of the present application is to prepare the progenitor cells from commonly available surgical to ‘waste’ has high therapeutic applicability, since no further invasive procedure need to be performed and no ethical concerns are raised.
In the method of present application, the tissue sample containing myometrium is treated with an enzyme to remove fibrous tissue. It is one-step enzyme treatment, and none of further steps such as filtration and gradient selection is needed. In one embodiment, the enzyme includes collagenase. The enzyme-treated tissue sample is then cultured in a serum-supplemented medium to obtain the progenitor cells. In one embodiment, the treated tissue sample is cultured in a complete medium supplemented by a serum and an antibiotic.
The present application also provides progenitor cells obtained from the above method. The progenitor cells are unique, differing from prior arts in terms of isolation method, differentiation capacity and cell surface marker expression profile.
The progenitor cells have negative expression of cell surface marker CD34, i.e. CD34(−). In one embodiment, the progenitor cells have positive expression of cell surface marker CD44, CD73, CD90, CD105, or any combination thereof. In one embodiment, the progenitor cells have negative expression of cell surface marker CD31, CD14, CD45, CD19, HLA-DR, Side Population (SidePop) or any combination thereof. In one preferred embodiment, the progenitor cells have positive expression of cell surface markers CD44, CD73, CD90 and CD105, and negative expression of cell surface markers CD31, CD34, CD14, CD45, CD19, HLA-DR and SidePop.
The progenitor cells of the present application can undergo osteogenesis, adipogenesis, chondrogenesis, and neurogenesis.
The progenitor cells of the present application possess strong immunomodulatory properties, which have suppressive effects on both CD4 and CD8 T lymphocytes.
The progenitor cells of the present application represent a new source of human stem cells which can be isolated without ethical concerns and in high volumes for wide clinical applicability. The clinical applications of the progenitor cells include, but is not limited to, degenerative diseases, ischemic diseases, a disease caused by abnormal immune response and the like.
Therefore, the present application further provides a method for treating a degenerative disease, an ischemic disease or a disease caused by abnormal immune response comprising administering the progenitor cells to a patient subjecting the disease.
In one embodiment, the degenerative disease includes, but is not limited to, Parkinson's disease, Alzheimer's disease, Huntington's disease, cerebral atrophy, cerebellar atrophy, schizophrenia and dementia.
In one embodiment, the ischemic disease includes, but is not limited to, stroke, cerebral apoplexy, cerebral hemorrhage, cerebral infarction, head trauma, vascular dementia and myocardial infarction.
In one embodiment, the disease caused by abnormal immune response includes, but is not limited to, autoimmune disease or graft rejection of organ transplantation. The autoimmune disease includes such as system lupus erythematosus, multiple sclerosis, rheumatoid arthritis, type 1 diabetes mellitus, coeliac disease, Sjögren's syndrome, Hashimoto's thyroiditis, Graves' disease, and idiopathic thrombocytopenic purpura.
In the method, the patient can be an mammal such as a rodent, primate, carnivora, artiodactyla and the like, preferably a primate. In one embodiment, the patient is a human.
Examples for preparing the progenitor cells are described hereinafter.
Methods & Materials
Cell Isolation & Culture
Uteri from hysterectomies for benign disease were obtained with informed consent approved by the institutional review board. The myometrium was dissected and separated from the endometrium and serosa, then digested with collagenase IV (Sigma-Aldrich) for 30 minutes. The samples and supernatant were then cultured in complete medium consisting of Dulbecco's modified Eagle's medium low glucose (Gibco-Invitrogen, Grand Island, USA) supplemented by 10% fetal bovine serum (FBS; selected lots, HyClone, Logan, Utah, USA), 100 U/ml penicillin, and 100 g/ml streptomycin (Sigma-Aldrich, St. Louis, Mo., USA). Cell cultures were maintained at 37° C. with a water-saturated atmosphere and 5% CO2. Medium was replaced one to two times every week, and when 80% confluent, cells were subcultured at 1:3 ratio.
Immunophenotyping
To detect surface antigens, aliquots of cells were washed with PBS containing 2% FBS after detachment with 0.25% trypsin/EDTA. All antibodies were purchased from BD Biosciences (Franklin Lakes, N.J.). Cells were stained with fluorescein isothiocyanate (FITC)- or phycoetrythrin (PE)-conjugated antibodies and compared with appropriate isotype controls. Flow cytometry analysis was performed using a FACSCalibur flow using CellQuest software (BD Biosciences) as we have previously reported (Yen, B. L., Huang, H. I., Chien, C. C., Jui, H. Y., Ko, B. S., Yao, M., Shun, C. T., Yen, M. L., Lee, M. C., and Chen, Y. C. (2005). Isolation of multipotent cells from human term placenta. Stem Cells 23, 3-9; Yen, B. L., Yen, M. L., Hsu, P. J., Liu, K. J., Wang, C. J., Bai, C. H., and Sytwu, H. K. (2013). Multipotent human mesenchymal stromal cells mediate expansion of myeloid-derived suppressor cells via hepatocyte growth factor/c-Met and STATS. Stem Cell Reports 1, 139-151). Sidepopulation cells were determined as previously reported (Ono et al. PNAS 2007) with the use of 50 μm verapamil to block efflux of the Hoechst 33342 dye (Sigma-Aldrich) (Goodell, M. A., Brose, K., Paradis, G., Conner, A. S., and Mulligan, R. C. (1996). Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo. J Exp Med 183, 1797-1806).
Differentiation Studies and Characterization
Differentiation into adipogenic, osteoblastic, and chondrogenic lineages was performed and characterized as we and others have previously reported (Liu, K. J., Wang, C. J., Chang, C. J., Hu, H. I., Hsu, P. J., Wu, Y. C., Bai, C. H., Sytwu, H. K., and Yen, B. L. (2011). Surface expression of HLA-G is involved in mediating immunomodulatory effects of placenta-derived multipotent cells (PDMCs) towards natural killer lymphocytes. Cell Transplant 20, 1721-1730; Pittenger, M. F., Mackay, A. M., Beck, S. C., Jaiswal, R. K., Douglas, R., Mosca, J. D., Moorman, M. A., Simonetti, D. W., Craig, S., and Marshak, D. R. (1999). Multilineage potential of adult human mesenchymal stem cells. Science 284, 143-147). Neurogenic differentiation was induced by standard methods; briefly, by culturing cells at low density (1000 cells/cm3), in serum-free medium with the addition of 0.5 μm retinoic acid (Sanchez-Ramos, J. R., Song, S., Kamath, S. G., Zigova, T., Willing, A., Cardozo-Pelaez, F., Stedeford, T., Chopp, M., and Sanberg, P. R. (2001). Expression of neural markers in human umbilical cord blood. Exp Neurol 171, 109-115), or in complete medium with the addition of 10 μm Y-27632 as we previously reported (Wang, C. H., Wu, C. C., Hsu, S. H., Liou, J. Y., Li, Y. W., Wu, K. K., Lai, Y. K., and Yen, B. L. (2013). The role of RhoA kinase inhibition in human placenta-derived multipotent cells on neural phenotype and cell survival. Biomaterials 34, 3223-3230 (“Wang et al.”). All reagents are from Sigma-Aldrich with the exception of TGF-β3 for chondrogenic differentiation, which was obtained from R&D Systems (Minneapolis, Minn.).
Immunofluorescence Staining
Immunofluorescence staining for neural characterization was performed as previously reported (Wang et al.). Briefly, cultured cells were fixed with 4% paraformaldehyde (PFA) (Sigma-Aldrich) for 10 minutes at room temperature and permeabilized with 0.1% Triton-X 100 (Sigma-Aldrich) for 10 minutes. Primary antibodies against the human antigens nestin and glial fibrillary acidic protein were purchased from Chemicon (Temecula, Calif.); for α-SMA were purchased from Sigma-Aldrich. Samples were first incubated with the primary antibodies at 4° C. overnight, then rinsed three times with PBS and incubated for 60 minutes at room temperature with FITC-conjugated secondary antibodies at a dilution of 1:100. All samples were stained with 4′,6-Diamidino-2-phenylindole (DAPI, 1:2000; Molecular Probes). Staining was visualized under a fluorescence microscope (Olympus, Tokyo, Japan).
Cell Proliferation Assessment
Cells were seeded initially at 1.5×104 cells/cm2 beginning from the 2nd passage (P2). Upon sub-confluent growth at a density of 80%, cells were trypsinized as usual and replated at the initial density. Growth curve were determined as we previously described (Ho et al.).
Reverse Transcription PCR (RTPCR)
RNA was isolated and RTPCR was performed as we previously reported (Ho et al.). Primers are as follows:
Progenitor/Stem Cell and Human Peripheral Blood Mononuclear Cells (PBMCs)/Lymphocyte Coculture Experiments
PBMC-related experiments were carried out similar to our previously described methods (Chang, C. J., Yen, M. L., Chen, Y. C., Chien, C. C., Huang, H. I., Bai, C. H., and Yen, B. L. (2006). Placenta-derived multipotent cells exhibit immunosuppressive properties that are enhanced in the presence of interferon-gamma Stem Cells 24, 2466-2477 (“Chang et al.”); Chen, P. M., Liu, K. J., Hsu, P. J., Wei, C. F., Bai, C. H., Ho, L. J., Sytwu, H. K., and Yen, B. L. (2014) Induction of immunomodulatory monocytes by human mesenchymal stem cell (MSC)-derived hepatocyte growth factor (HGF) through ERK1/2. J Leukoc Biol 95, 295-303 (“Chen et al.”)). Briefly, human PBMCs were isolated from the buffy coat of healthy donor blood samples (Taiwan Blood Services Foundation, Taipei Blood Center, Taipei, Taiwan) obtained with informed consent approved according to the procedures of the institutional review board and cultured as previously reported. Isolated PBMCs were first stained with carboxyfluorescein succinimidyl ester (CFSE; Gibco-Invitrogen)—a green fluorescence dye—to track for cell division after stimulation with phytoagglutinin (PHA; Sigma-Aldrich) or anti-CD3/28 beads (a-CD3/28; Dynabeads) which more specifically stimulates T lymphocytes. Progenitor/stem cells (MDMPs or BMMSCs) were plated at 3.5×104 cells per well in 6-well plates, and incubated at 37° C. for 24 hours prior to co-culture with stimulated PBMCs (1×105). After 3 days further of culture, cells were harvested and evaluated by flow cytometric analysis for CFSE staining intensity or various protein (surface marker, cytokine, transcription factor, etc.) expression.
Mouse In Vivo Experiments
All animal work was performed in accordance with protocols approved by the institutional Animal Care and Use Committee. Wild-type C57BL/6J mice were purchased from the National Laboratory Animal Center of Taiwan (Taipei, Taiwan). Induction of proinflammatory leukocytes in vivo was performed similarly as previously reported (Shi, G., Vistica, B. P., Nugent, L. F., Tan, C., Wawrousek, E. F., Klinman, D. M., and Gery, I. (2013). Differential involvement of Th1 and Th17 in pathogenic autoimmune processes triggered by different TLR ligands. J Immunol 191, 415-423). Briefly, lipopolysaccharide (LPS; 100 μg, Escherichia coli 00041:B4; Sigma-Aldrich) was injected intraperitoneally into 8- to 12-week-old mice, followed 2 hours later by transfer of either MDMPs or BMMSCs (1×105 cells/mouse). Mice were sacrificed on day 3 with harvesting of leukocytes from the spleen and regional lymph nodes for assessment of Th1 cells and Tregs as previously reported (Chen et al; Chang et al).
Results
Characterization of Multipotent Progenitors From the Human Uterine Corpus
Myometrial-derived multipotent progenitors (MDMPs) were isolated from posthysterectomy specimens of benign diagnosis. These MDMPs are highly roliferative, even as compared to somatic progenitors isolated from adipose tissue, which is currently a popular source for isolation of human therapeutic progenitors (
MDMPs Possess Multilineage Potential
The differentiation potential of MDMPs were then assessed. It was found that MDMPs can differentiate into multiple cell lineages, including osteogenic, hondrogenic, adipogenic, and neurogenic lineages (
MDMPs Possess More Significant In Vitro and In Vivo Immunomodulatory Capacity Than BMMSCs
Increasing evidence show that inflammation is involved in multiple disease entities previously not thought to involve such processes, and this includes such epidemiologically prominent diseases including neurodegenerative diseases, ischemic diseases and cancer. The uterus has been postulated to have a unique immunological milieu (Moffett-King, A. (2002). Natural killer cells and pregnancy. Nat Rev Immunol 2, 656-663). Thus, whether MDMPs possess immunomodulatory effects was evaluated. To assess whether MDMPs are immunosuppressive, it was performed mixed lymphocyte reactions using stimulated human peripheral blood monoclear cells (PBMCs) and co-cultured either MDMPs or BMMSCs—a known immunomodulatory type of stem/progenitor cells—simultaneously. It was found that coculture of MDMPs not only suppressed the proliferation of PBMCs stimulated with either phytoagglutinin or anti-CD3/28 beads, but can do so more potently than BMMSCs can be significantly suppressed when MDMPs are co-cultured (
Comparison Between the MDMPs and the Prior Arts
Referring to Table 1, summary of the comparison are shown.
Ono et al. (Ono et al, PNAS 2007) disclosed that the MyoSPs were isolated by incubating myometrial tissue in medium with minimal enzyme addition (0.02%) for 4-16 hours with subsequent filtration (2 times) and gradient selection (Ficollpaque), then the tissue was further trypsinized.
Galvez et al., In Vivo 2009, WO2010/057965 and WO2011/042547A1 disclose that AMPs were isolated by incubating the myometrial tissue in serum-free medium, and selecting only fragments with small vessels for further culturing. The method likely selects endothelial-like cells, and the results showing high CD31(+) percentage of these isolated AMPs (>99.7% for mouse AMPs and >90% for human AMPs) can be explained accordingly.
In the method of the present application, a one-step of enzymatic treatment is applied for less than 1 hour without filtration or gradient selection, with culturing in serum-supplemented medium. In addition, there is no need to use portions with vessels for further culturing in the present application.
MyoSPs were characterized by the ability to efflux Hoechst 33342 dye, which show up on flow cytometric analysis as a “side population,” which the present application does not yield (see
In addition, MyoSPs are unable to differentiate into chondrogenic cells, nor reported to differentiate into neurogenic cells, as MDMPs can Immunomodulatory capacity of MyoSPs are not discussed by Ono et al., but MyoSPs may unlikely to have this capacity since they are CD34(+) and CD31(+), indicating a hematopoietic background and likely immunogenic.
Only mouse AMPs but not human AMPs were tested for differentiation capacity. The mouse AMPs possessed osteogenesis, adipogenesis and neurogenesis, but no chondrogenesis was reported Immunomodulatory capacity of AMPs are not discussed in the above disclosures, but AMPs could possibly be immunogenic due to being HLA-DR (+) at baseline.
Obviously, MDMPs possesses distinct characteristics from MyoSPs and AMPs.
While the present invention is disclosed by reference to the preferred embodiments and examples detailed above, it is to be understood that these examples are intended in an illustrative rather than in a limiting sense. It is contemplated that modifications and combinations will readily occur to those skilled in the art, which modifications and combinations will be within the spirit of the invention and the scope of the following claims and its equivalent systems and methods.
This application claims priority of U.S. provisional application No. filed 62/052,088 on Sep. 18, 2014.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2015/050418 | 9/16/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62052088 | Sep 2014 | US |
