TECHNICAL FIELD

The invention relates to methods for generating multipotent mammary stem cells from isolated and cultured human breast luminal cells. The invention also relates to appropriate culture media used by the methods disclosed; multipotent mammary stem cells, cultures of the multipotent stem cells, differentiated cells derived from the culture multipotent stem cells isolated by the methods disclosed and therapeutic uses for those cells.

BACKGROUND OF THE INVENTION

Mammary stem cells are multipotent adult stem cells that reside in the mammary gland, have self-renewal properties by dividing and differentiating into the specialized mammary epithelial cells. Mammary stem cells contribute to the development of the mammary gland during puberty and are responsible for the significant expansion during pregnancy. Mammary stem cells give rise to mature epithelium of the mammary gland which is composed of two cellular lineages: The luminal lineage and the myoepithelial lineage via a series of lineage-restricted intermediates.

In human breast tissue, the observation of identical chromosomal alterations in contiguous regions of human breast epithelium has implied the presence of mammary stem cells (Deng et al., Loss of heterozygosity in normal tissue adjacent to breast carcinomas. Science 1996, 274:2057-2059; Lakhani et al., Detection of allelic imbalance indicates that a proportion of mammary hyperplasia of usual type are clonal, neoplastic proliferations. Lab Invest 1996, 74:129-135). In the recent ten years, much progress has been made in the identification of signaling pathways that function in mammary stem cells to control self-renewal, lineage commitment and epithelial differentiation in the normal mammary gland. Meanwhile, the same signaling pathways that control physiological mammary development and homeostasis are also often found deregulated in breast cancer (Ercan et al., Mammary Development and Breast Cancer: The Role of Stem Cells, Curr Mol Med. 2011 June; 11(4): 270-285).

In recent studies, breast cancer was found to be the most common cancer in women worldwide, with nearly 1.7 million new cases diagnosed in 2012 (Internet, World Cancer Research Fund International, Worldwide Website: wcrf.org/int/cancer-facts-figures, accessed on Jun. 9, 2016). Even though treatment for breast cancer has been significantly improved and the survival rate has been increased, this disease is still a big health problem for human being. Therefore, the research concerning the mammary gland stem cell and breast cancer stem cell might provide motivations to find the complete treatment of this deadly disease (Kordon E C, Smith G H, an entire functional mammary gland may comprise the progeny from a single cell. Development. 1998; 125:1921-30). Researchers believe that once the stem cell is isolated, the signaling pathways that regulate its proliferation, self-renewal, survival and differentiation will be illustrated, and this might shed light on the mechanisms of breast cancer formation, therefore leading to a better therapeutic treatment (Wang R H, the new portrait of mammary gland stem cells. Int J Biol Sci 2006; 2(4):186-187).

In addition, the research on mammary stem cell may provide an active solution for organ transplantation. With respect to allotransplantation, the common problem is that there is no enough organ donations. With respect to xenotransplantation, it also raises many novel medical issues. For example, xenotransplantation does not overcome the problem of tissue incompatibility and furthermore, there is evidence that damaging viruses cross species barriers.

Recent success at transplanting through stem cells have provided new clinical tools to reconstitute and/or supplement bone marrow after myeloablation due to disease or damages seduced by the harmful environment. It was also reported that a single mammary stem cell can reconstitute a complete mammary gland in vivo (Shackleton M., et al., Nature. 2006 Jan. 5; 439(7072):84-8). The application of stem cells in tissue engineering, gene therapy delivery and cell therapeutics is also advancing rapidly.

However, a basic problem existed prior to the present invention. It is difficult to obtain sufficient quantities and populations of human stem cells which are capable of differentiating into most cell types. The isolation of normally occurring populations of stem cells in adult tissues is known to be technically complicated and costly due to the limited quantity found in human tissue. The harvest of these stem cells from embryos or fetal tissue is morally questionable, if not forbidden anyway for religious, legal and ethical reasons.

There is therefore a need for developing methods for generating multipotent mammary stem cells.

There is also a need to provide multipotent mammary stem cells, cultures of the multipotent stem cells, differentiated cells, tissues and also organs derived from the cultured multipotent stem cells, for their applicability in tissue engineering and cell therapeutics. This will also help scientists and researchers to gain a better understanding of mammary stem cells, and will ultimately lead to better therapies including breast cancer therapies.

There is also a further need for a source of cells that are transplantable to in vivo tissues/organs in order to replace diseased or damaged tissues/organs.

SUMMARY OF THE INVENTION

In one aspect the present invention relates to a method for generating multipotent mammary stem cells comprising the steps:a) isolating and growing primary breast luminal cells in-vitro;b) treating the primary breast luminal cells with either a conditioned medium from active breast stromal fibroblasts or with interleukin-8 (IL-8) in serum-free medium;c) continuing the growth of cells until the formation of cellular aggregates, wherein preferably such growth is done in a breast luminal-cell-specific medium; andd) transferring the cells to a stem cell growth medium and growing them further until the formation of mammospheres.

