Myogenic transdifferentiation of menstrual blood-derived cells

Summary

Cells with myogenic potential are present in many tissues, and these cells readily form skeletal muscle in culture. We here focus on menstrual blood as another cell source for regenerative medicine. Menstrual blood-derived cells have high replicative ability, similar to progenitors or stem cells, and transdifferentiate or meta-differentiate into myocytes in vitro at unexpectedly high frequencies. This unique phenotype can be explained by histological and embryological aspects of the endometrium. The remarkable myogenic capability of these cells enables us to “rescue” dystrophied myocytes of the mdx model of Duchenne muscular dystrophy through cell fusion and transdifferentiation. Endometrial cells supplied as a form of menstrual blood-tissue mixture can be used for cell-based therapy in addition to a place for embryo implantation.

Introduction

Mesenchymal stem cells and mesenchymal cells are attracting a great deal of attention, as they represent a valuable source of cells for use in regenerative medicine, as well as offering an excellent model of cell differentiation in biology (1, 2). To date, investigators have demonstrated that mesenchymal stem cells per se can be recovered from bone marrow, fat, muscle, placenta, umbilical cord, cord blood and skin, and have the capacity to differentiate into a variety of specific cell types (3–5). In addition to these tissues, menstrual blood is one of the best sources of mesenchymal stem cells with remarkable myogenic potential (6).

Menstrual Blood

Menstrual cycles occur exclusively in humans and other great apes. Menstruation is the phase of the menstrual cycle in which the endometrium is shed. Normal, regular menstruation lasts for a few days (usually 3 to 5 days). The average blood loss during menstruation is 35 ml. To culture menstrual blood-derived cells, menstrual blood is usually obtained on the first day of menstrual bleeding, because menstruation on the first day contains many cells and tissues mixed with the blood due to shedding of the endometrium (Fig. 1). The menstrual cycle is under the control of the hormone system. During the proliferative phase, the endometrium thickens, stimulated by gradually increasing amounts of estrogen. Follicles in the ovary begin developing under the influence of a complex interplay of hormones and, after several days, one or occasionally two follicles become dominant. The dominant follicle releases an ovum or egg in an event called ovulation. After ovulation, the remains of the dominant follicle in the ovary become the corpus luteum; this body has the primary function of producing large amounts of progesterone. Under the influence of progesterone, the endometrium changes to prepare for potential implantation of an embryo to establish a pregnancy. If implantation does not occur within approximately two weeks, the corpus luteum will die, causing sharp drops in the levels of both progesterone and estrogen. These hormone drops cause the uterus to shed the endometrium in a process termed menstruation.

Figure 1.

Myogenic transdifferentiation of menstrual blood-derived cells. Menstrual blood, which is usually obtained on the first day of menstrual bleeding, contains many cells and tissues mixed with the blood due to shedding of the endometrium. The remarkable myogenic capability of cultured menstrual blood-derived cells enables us to “rescue” dystrophied myocytes of the mdx model of Duchenne muscular dystrophy through cell fusion and transdifferentiation.

Structure of endometrium

Menstrual blood contains cells or tissues from the functional layer of the endometrium. This layer is built up after the end of menstruation during the first part of the previous menstrual cycle. Proliferation is induced by progesterone (proliferative phase of the menstrual cycle), and later increased by the progestrone from the corpus luteum (secretory phase). In the absence of progesterone, the arteries supplying blood to the functional layer constrict, so that cells in that layer become ischemic and die, leading to menstruation. In contrast, the basal layer, adjacent to the myometrium and below the functional layer, is not shed during the menstrual cycle. Histologically, the functional endometrium consists of a single layer of columnar epithelium, resting on a layer of connective tissue which varies in thickness according to hormonal influences – the stroma. Simple tubular uterine glands reach from the endometrial surface through to the base of the stroma, which also carries a rich blood supply through spiral arteries.

Myogenic potential of menstrual blood-derived cells

Human menstrual blood-derived cells (Fig. 1) possess high self-renewal capacity over at least 25 Population Doublings (PDs) (9 passages) for more than 60 days, and stop dividing before 30 PDs. This cessation of cell division is probably due to replicative senescence or shortening of telomere length. It is important that these cells be obtained from younger patients, because they will have a longer life span than cells harvested from older donors. Menstrual blood-derived cells have high replicative ability, similar to progenitors or stem cells that display long-term self-renewal capacity, and have a much higher growth rate than either fetal cells (7) or marrow-derived stromal cells (8). In spite of a high growth rate, the cell life span of menstrual blood cells is relatively short when compared with human fetal cells (7), and this shorter cell life span may be attributed to the shorter telomere length of adult cells, i.e., endometrial stromal cells, at the start of cell cultivation. Menstrual blood-derived cells efficiently transdifferentiate into myoblasts/myocytes, and fuse to myoblasts in vitro and in vivo by appropriate induction (6), and this myogenic potential of menstrual blood-derived cells is much greater than expected. Constitutive expression of MyoD, desmin, and myogenin, markers for skeletal myogenic differentiation in menstrual blood-derived cells, implies either that most of these cells are myogenic progenitors, or that these cells have myogenic potential. Myogenic differentiation of menstrual blood-derived cells reminds us of “mixed Müllerian tumors of the endometrium”. This tumor contains both epithelial and stromal elements, and the stromal elements tend to differentiate into a variety of mesodermal components, including striated muscle cells. The caudal portion of the Müllerian ducts matures into the uterus and upper vagina in human embryogenesis. Divergent differentiation, including myogenesis of menstrual blood-derived cells (or endometrium-derived cells), may be a reflection of the plasticity of primitive Müllerian cells. Menstrual blood-derived cells show at least two morphologically different cell groups: small spindle-like cells and large stick-like cells (Fig. 1). Surface markers of menstrual blood-derived cells are evaluated by flow cytometric analysis. Menstrual blood-derived cells are positive for CD13, CD29, CD44, CD54, CD55, CD59, CD73, CD90, and CD105, implying that proliferated and propagated cells express mesenchymal cell-related cell-surface markers. Menstrual blood-derived adherent cells are positive for CD105. Menstrual blood-derived cells express neither hematopoietic lineage markers, such as CD34, nor monocyte-macrophage antigens such as CD14 (a marker for macrophage and dendritic cells), and CD45 (leukocyte common antigen). The lack of expression of CD14, CD34, or CD45 suggests that menstrual blood-derived cell culture is depleted of hematopoietic cells. The cells are also negative for the expressions of CD31 (PECAM-1), CD50, c-kit, and CD133. The cell population is positive for HLA-ABC, but not for HLA-DR. Almost all cells derived from the endometrium are of mesenchymal or stromal origin.

Cell fusion between menstrual blood cells and myoblasts

Menstrual blood cells after myogenic differentiation become able to fuse with myoblasts, and confer dystrophin on dystrophied myocytes. The acquisition or recovery of dystrophin expression in dystrophic muscle is attributed to two different mechanisms: a) myogenic differentiation of implanted or transplanted cells or b) cell fusion of implanted or transplanted cells with host muscle cells. Recovery of dystrophin-positive cells is explained by muscular differentiation of implanted marrow stromal cells and adipocytes (9, 10). In contrast, implantation of normal myoblasts into dystrophin-deficient muscle can create a reservoir of normal myoblasts that are capable of fusing with dystrophic muscle fibers and restoring dystrophin (11). Cellular fusion between adipocytes and dystrophied myocytes is involved in dystrophin expression of Duchenne muscular dystrophy myofibers (10). Dystrophin expression in dystrophied myocytes is detected only when cells of mesenchymal origin are implanted, and is correlated with a high frequency of cell fusion between donor cells and host myocytes (6).

Menstrual blood as a novel source of cell-based therapy

Human menstrual blood-derived cells offer several important advantages. First, human primary menstrual blood-derived cells are obtained by a simple, safe, and painless procedure, and can be expanded efficiently in vitro. In contrast, the isolation of mesenchymal stem cells/mesenchymal cells from other sources, such as bone marrow and adipose tissue, is accompanied by a painful and complicated operation. Second, the transplantation of menstrual blood-derived cells should encounter fewer ethical problems because the use of these cells avoids the embryonic stem cell controversy. Third, autologous transplantation of menstrual blood-derived cells or transplantation of these cells with the same HLA subtype from a healthy donor should minimize the risk of rejection. Because general populations of menstrual blood-derived cells are adherent, functional skeletal muscle cells can be obtained within a reasonable time on a therapeutic scale. Duchenne muscular dystrophy is a devastating X-linked muscle disease characterized by progressive muscle weakness attributable to a lack of dystrophin expression at the sarcolemma of muscle fibers (10, 11), and there are no effective therapeutic approaches for muscular dystrophy at present. Efficient fusion systems of menstrual blood-derived cells with host dystrophic myocytes may contribute substantially to a major advance toward eventual cell-based therapies for muscle injury or chronic muscular disease.