Methods for generating multipotent stem cells from tonsillar biopsies

#Multipotent progenitor cells (MPCs) can give rise to several types of specialized cells (Pittenger et al., (1999) Science, 284:143-147). These fibroblast-like cells were first identified and isolated from the bone marrow (BM) and spleen (Friedenstein et al., (1970) Cell Tissue Kinet, 3:393-403). Presently, many other tissues such as placenta, amniotic fluid, umbilical cord, adipose tissue, tonsils and endometrium have been identified as sources of MPCs (Baksh et al., (2007) Stem Cells, 25:1384-92; De Coppi et al., (2007) Nat Biotechnol, 25:100-106; Fukuchi et al., (2004) Stem Cells, 22:649-658; Gargett et al., (2009) Biology of Reproduction, 80:1136-1145; Lee et al., (2016) Cell Physiol Biochem, 38:83-93; Park et al., (2015) Sci Rep, 5:8616; Ryu et al., (2012) Cytotherapy, 14:1193-1202; Zuk et al., (2002) Molecular Biology of the Cell, 13:4279-4295). Nevertheless, the phenotype and differentiation potency of MPCs vary with respect to the tissue source from which they are isolated and the harvesting procedure (Heidari et al., (2013) Avicenna J Med Biotechnol, 5:104-117; Heo et al., (2016) Int J Mol Med, 37:115-125; Tan et al., (2012) Tissue Eng Part A, 18:840-851). MPCs possess the potential to differentiate into multiple cell types including adipocytes (fat), chondrocytes (cartilage), and osteoblasts (bone) (Heino and Hentunen. (2008) Curr Stem Cell Res Ther, 3:131-145). Characterization of MPCs includes adherence to standard tissue culture plastic, expression of various surface antigens, and in vitro differentiation potential (Dominici et al., (2006) Cytotherapy, 8:315-317). Like other types of adult stem cells, MPCs remain quiescent (non-dividing) for long periods. When activated, MPCs divide and differentiate to replace injured cells and secrete factors to prevent inflammation and promote tissue repair (Murphy et al., (2013). Exp Mol Med, 45:e54).

#Cellular therapies hold a great potential for the cure of a wide range of diseases and provide enhanced treatment modalities including immunomodulatory therapies, tissue regeneration and cancer therapies. Using MPCs is an attractive approach for cell therapy as it avoids the ethical and practical issues of embryonic and fetal-derived stem cells (Ding et al., (2011) Cell Transplant, 20:5-14). Importantly, MPCs migrate into sites of tissue injury and have strong immunosuppressive properties that can be exploited for successful autologous and for allogenic transplantation without the need of complex and harmful anti-rejection medication protocols (Murphy et al., (2013) Exp Mol Med, 45:e54; Le Blanc and Ringden, (2005) Biol Blood Marrow Transplant, 11:321-34). Currently, many clinical trials are testing MPCs obtained from different tissue sources for the treatment of numerous conditions such as autoimmunity, heart disease, bone and cartilage disease, cancer, neuropathologies and gastrointestinal diseases (Kim and Cho, (2013) Korean J Intern Med, 28:387-402). Human MPCs are also equally used in drug discovery applications as replacements for primary cells and animal models for initial toxicity and screening of new compounds (Scanu et al., (2011), Toxicology in Vitro, 25:1989-1995).

#MPCs reside on blood vessels (Crisan et al., (2008) Cell Stem Cell, 3:301-313; Bell et al., (2010) Neuron, 68:409-427; Murray et al., (2014) Cell Mol Life Sci, 71:1353-1374) and therefore, the more vascularized the tissue is, the more rich the tissue is with MPCs. Nevertheless, the tissue source and the cells' derivation procedure can affect the abundance, phenotype and differentiation potency of MPCs (Baksh et al., (2007) Stem Cells, 25:1384-92; Heidari et al., (2013) Avicenna J Med Biotechnol, 5:104-117; Heo et al., (2016) Int J Mol Med, 37:115-125; Tan et al., (2012) Tissue Eng Part A, 18:840-851; De Bari et al., (2008) Arthritis Rheum, 58:240-250). Bone marrow (BM) has been one of the major sources of MPCs. However, the derivation of stem cells from patients and healthy donors is not always possible. Bone marrow extraction requires hospitalization and leads to considerable donor morbidity, including pain and bleeding, and other complication such as infection and risk for viral exposure (Buckner et al., (1984) Blood, 64:630-634; Nishimori et al., (2002) Blood, 99:1995-2001). Therefore, efforts have been made to find alternative sources of MPCs for cell therapies. Another current major source for MPC derivation is adipose tissue. However, 100 ml of tissue and blood collected from lipoaspiarates provides approximately 105 cells (Francis et al., (2010) Organogenesis, 6:11-4). This is likely due to the mechanical trauma to the tissue incurred during the liposuction procedure, leading to a low yield and reduced cell viability (Zuk et al., (2002) Mol Biol Cell, 13:4279-4295). Yet, in order to achieve enough stem cells in the scale needed for clinical purposes, a large quantity of starting tissue material is needed which may result in considerable risk and donor morbidity.

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#FIG. 1 depicts exemplary experimental results demonstrating a schematic workflow of tonsillar multipotent progenitor cell (T-MPC) isolation from human tonsillar biopsy. T-MPCs can be expanded in culture and differentiated into various lineages including: smooth muscle cells, osteoblasts (bone cells), chondrocytes (cartilage), adipocytes (fat cells) and neurons.

#FIG. 2, comprising FIG. 2A through FIG. 2D, depicts exemplary experimental results demonstrating characterization of T-MPCs by immunostaining and flow cytometry. FIG. 2A depicts exemplary experimental results demonstrating that an analysis of co-staining demonstrates that the T-MPCs are CD44 and CD90, CD73, and CD105 positive and CD19 and HLA-DR negative. n=14 donors in triplicates. Bm-MPC=bone marrow-derived mesenchymal progenitor cell, T-MPC=tonsillar mesenchymal progenitor cells. FIG. 2B depicts exemplary experimental results demonstrating that a fraction of the T-MPCs express the pluripotency related marker SSEA4. FIG. 2C depicts exemplary experimental results demonstrating that a fraction of the T-MPCs express the pluripotency related marker TRA-1-80. FIG. 2D depicts exemplary experimental results demonstrating that a fraction of the T-MPCs express the pluripotency related marker TRA-1-60.

#The present invention relates to the discovery of a method for isolating multipotent progenitor cells (MPCs) from small amounts of tonsillar tissue (T-MPCs) in a manner that retains the ability of the cells to proliferate and differentiate into multiple cell lineages including, but not limited to osteoblasts, chondrocytes, and adipocytes.

#Described herein are methods of identifying and/or isolating T-MPCs. These T-MPCs can be distinguished by expression of cell surface markers such as CD44, CD90, CD73, CD105, TRA-1-80, and TRA-1-60.

#Also described herein are therapeutic methods using the T-MPCs. In various embodiments, the T-MPCs or differentiated cells derived therefrom can be used to treat or prevent blood and lymph conditions, cancers and other neoplasms, digestive system diseases, diseases and abnormalities at or before birth, ear, nose, and throat diseases, eye diseases, gland and hormone-related diseases, heart and blood diseases, immune system diseases, mouth and tooth diseases, muscle, bone, and cartilage diseases, nervous system diseases, nutritional and metabolic diseases, occupational diseases, respiratory tract (lung and bronchial) diseases, skin and connective tissue diseases, substance-related disorders, symptoms and general pathology, urinary tract, sex organ, and pregnancy-related conditions, viral diseases, and wounds and injuries.