Role of tumor suppressor genes in the cancer-associated reprogramming of human induced pluripotent stem cells.

Because of their pluripotent characteristics, human induced pluripotent stem cells (iPSCs) possess great potential for therapeutic application and for the study of degenerative disorders. These cells are generated from normal somatic cells, multipotent stem cells, or cancer cells. They express embryonic stem cell markers, such as OCT4, SOX2, NANOG, SSEA-3, SSEA-4, and REX1, and can differentiate into all adult tissue types, both in vitro and in vivo. However, some of the pluripotency-promoting factors have been implicated in tumorigenesis. Here, we describe the merits of tumor suppresser genes as reprogramming factors for the generation of iPSCs without tumorigenic activity. The initial step of reprogramming is induction of the exogenous pluripotent factors to generate the oxidative stress that leads to senescence by DNA damage and metabolic stresses, thus inducing the expression of tumor suppressor genes such as p21CIP1 and p16INK4a through the activation of p53 to be the pre-induced pluripotent stem cells (pre-iPSCs). The later stage includes overcoming the barrier of reprogramming-induced senescence or cell-cycle arrest by shutting off the function of these tumor suppressor genes, followed by the induction of endogenous stemness genes for the full commitment of iPSCs (full-iPSCs). Thus, the reactive oxygen species (ROS) produced by oxidative stress might be critical for the induction of endogenous reprogramming-factor genes via epigenetic changes or antioxidant reactions. We also discuss the critical role of tumor suppressor genes in the evaluation of the tumorigenicity of human cancer cell-derived pluripotent stem cells, and describe how to overcome their tumorigenic properties for application in stem cell therapy in the field of regenerative medicine.

Autocrine and paracrine mechanisms regulating primordial germ cell proliferation.

Although several mitogens and survival factors have been previously shown to act on primordial germ cells (PGCs) in culture, it is not clear whether they are responsible for controlling proliferation of PGCs in the embryo. We show here that during their migratory phase, PGCs do not express FGF-4, FGF-8, or FGF-17, but these FGFs are expressed by neighboring cells. Thus, any FGF action on migrating PGCs would appear to be through a paracrine mechanism. We found that after entering into the gonads, PGCs start to express FGF-4 and FGF-8. On this basis, we hypothesize that FGF signaling is involved in both a paracrine manner in initiating PGC proliferation during their migration and an autocrine manner in sustaining PGC proliferation after their arrival in the gonads. We then studied the role of soluble stem cell factor (SCF), which acts as a survival factor or a mitogen in culture, to determine whether it interacts with FGFs. We found that SCF has a complex effect on PGC proliferation. On one hand, soluble SCF promoted PGC proliferation synergistically with FGF in the absence of membrane-bound SCF. Conversely, soluble SCF inhibited FGF-stimulated proliferation of PGCs in the presence of membrane-bound SCF. We account for these findings in a model involving regulation of PGC proliferation, in which SCF modulates the response to FGFs.

A combination of Buffalo rat liver cell-conditioned medium, forskolin and membrane-bound stem cell factor stimulates rapid proliferation of mouse primordial germ cells in vitro similar to that in vivo.

Recent studies have shown that stem cell factor (SCF), leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and the enhancement of cAMP levels increase proliferation and survival of mouse primordial germ cells (PGC) in vitro. Even after the addition of these factors, however, it is still not possible to obtain proliferation of PGC at a rapid rate similar to that in vivo, suggesting the presenge of other growth factor(s) in vivo. We previously reported that tumor necrosis factor-α stimulates proliferation of PGC at earlier migration stages. We now show that the use of SI/SI4-m220 feeder cells and the addition of a medium conditioned with Buffalo rat liver cells and forskolin to the culture medium stimulate PGC obtained from 8.5 days post coitum embryos to proliferate in culture at a rate comparable to that in vivo. Under such conditions, proliferation of PGC continued several days past the timing of growth arrest in vivo; however, it did stop afterwards. Such proliferating PGC continue to express c-kit and Oct-3 proteins. The characteristics of the culture medium and the requirement of feeder cells were different from those for embryonic stem (ES) cells, suggesting that these rapidly proliferated PGC are not transformed into ES-like EG cells.

Mouse Offspring Derived from Fetal Ovaries or Reaggregates Which were Cultured and Transplanted into Adult Females.

Primordial germ cells (PGCs) and gonia could be promising novel targets and vehicles for manipulation of the mammalian germ line. To make such manipulation a practical possibility, PGCs or gonia must be allowed to produce gametes and offspring after they were isolated from embryos and manipulated in culture. As the first step to develop such research strategy, we obtained offspring from mouse oogonia which were isolated from embryonic ovaries and cultured as dispersed cells before transplantation into female mice as reaggregates.

Minute protrusions of the cuticle: Fine surface structures of the tunic in ascidians.

The fine structure of the cuticular surface of the tunic was studied by scanning and transmission electron microscopy in 25 species belonging to 9 families of ascidians. The cuticular surface is ornamented with numerous minute protrusions in some species, but not in others. The minute protrusions are usually papillate in shape, and less than ∼0.05 µm high (in some species of the families Polyclinidae and Polycitoridae), or ∼0.1 µm high (in all species of Botryllidae, all colonial species of Styelidae and one solitary species of Pyuridae). In Clavelina miniata (Polycitoridae), the tunic is provided with parallel ridges on the surface of which papillate protrusions are distributed. The minute protrusions of Halocynthia roretzi (Pyuridae) are irregularly shaped, giving an appearance of paving stones. No protrusions are found in the families Didemnidae, Cionidae, Perophoridae, and Ascidiidae, and in one solitary species of Styelidae. At least in some colonial species, the density of the minute protrusions is lower at the edge of the colony than elsewhere. It is very probable that minute protrusions are found shortly after the secretion of tunic, growing larger both in size and in number as the tunic becomes older.

Tunic cords, glomerulocytes, and eosinophilic bodies in a styelid ascidian, Polyandrocarpa misakiensis.

Some unusual specialized structures of the tunic and epidermis in Polyandrocarpa misakiensis were studied histologically, histochemically, and electron microscopically. The tunic is leathery, containing fibers. Dorsal epidermal cells show cytoplasmic bulges at their apical ends, suggesting formation of tunic by the epidermis. Both siphons are characterized by a well-developed velum. In the dorsal mantle, especially around the siphons, numerous elongate cord-like structures of the tunic (10-30 µm in diameter) extend into the hemocoel. Each tunic cord originates from the extra-epidermal tunic, ends distally in a spade-shaped swelling, and is covered by flattened epidermal cells. The tunic cords probably act as connectors between tunic and mantle. Glomerulocytes, a kind of blood cell, are distributed throughout the hemocoel. They are disc-shaped (12-13 µm in diameter, 3 µm thick), and are characterized by a concentric fiber structure in the cytoplasm. The intracellular fibers are similar to the tunic fibers both morphologically and histochemically. The glomerulocytes appear to be derived from epidermal cells; their differentiation occurs in all regions of the epidermis. The function of glomerulocytes is unknown. Small club-shaped bodies which are intensely eosinophilic are distributed here and there in the dorsal epidermis. These eosinophilic bodies are usually located individually in epidermal pockets, and are protruded into the hemocoel. The eosinophilic body is proteinaceous and PAS-positive; it may be a secretory product of the covering epidermis. No physiological role of the eosinophilic body is yet clear. During blasto-genesis, incipient tunic cords, glomerulocytes, and eosinophilic bodies appear almost simultaneously at a stage shortly before the opening of siphons.

Formation of the Primitive Streak and Mesoderm Cells in Mouse Embryos-Detailed Scanning Electron Microscopical Study: (primitive streak/cell migration/extracellular matrix/mouse gastrulation/scanning electron microscopy).

We describe morphological events of the mammalian gastrulation in pre- to middle-primitive-streakstage mouse embryos by using scanning electron microscopy. The first sign of the ingression of the mesodermal cells was disruption of the epithelial structure of ectoderm and the underlying basal lamina, thus forming a semicircular area of the presumptive primitive streak. Then, cells at periphery of the semicircular region spread on the basal lamina by extending many filopodia to it. The majority of the migrating cells formed a loosely arranged cell sheet. We found solitary cells and isolated small groups of cells migrating away from the periphery of the cell sheet. These cells were well spread on the basal lamina, and had large cell processes and many filopodia in the direction of cell migration. Filopodia of these cells were attached to the basal lamina or a meshwork of the extracellular fibrils. These observations suggest that the extracellular matrix serves as the substratum for cell adhesion and migration, and plays an important role in the mammalian gastrulation.

Laminin Fibrils in Newt Gastrulae Visualized by the Immunofluorescent Staining: (gastrulation/laminin/immunofluorescent staining/newt/amphibian).

An anastomosing network of extracellular fibrils on the inner surface of the ectoderm layer of amphibian gastrulae has been shown to provide an adequate substratum for attachment and migration by the mesodermal cells. These fibrils contain fibronectin as shown by immunostaining at the light and electron microscope levels. Now we report the presence of laminin, another cell adhesion glycoprotein, as a fibrillar network on the inner surface of the ectoderm layer in gastrulae of the Japanese newt (Cynops pyrrhogaster), but its absence on the blastula ectoderm layer, by the immunofluorescent staining using an antiserum specific for mouse laminin. The same antiserum was shown to stain basement membranes of adult newt organs as expected.