Makoto Goda

Unique multipotent cells in adult human mesenchymal cell populations

We found adult human stem cells that can generate, from a single cell, cells with the characteristics of the three germ layers. The cells are stress-tolerant and can be isolated from cultured skin fibroblasts or bone marrow stromal cells, or directly from bone marrow aspirates. These cells can self-renew; form characteristic cell clusters in suspension culture that express a set of genes associated with pluripotency; and can differentiate into endodermal, ectodermal, and mesodermal cells both in vitro and in vivo. When transplanted into immunodeficient mice by local or i.v. injection, the cells integrated into damaged skin, muscle, or liver and differentiated into cytokeratin 14-, dystrophin-, or albumin-positive cells in the respective tissues. Furthermore, they can be efficiently isolated as SSEA-3(+) cells. Unlike authentic ES cells, their proliferation activity is not very high and they do not form teratomas in immunodeficient mouse testes. Thus, nontumorigenic stem cells with the ability to generate the multiple cell types of the three germ layers can be obtained through easily accessible adult human mesenchymal cells without introducing exogenous genes. These unique cells will be beneficial for cell-based therapy and biomedical research.

Multilineage-differentiating stress-enduring (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts

The stochastic and elite models have been proposed for the mechanism of induced pluripotent stem (iPS) cell generation. In this study we report a system that supports the elite model. We previously identified multilineage-differentiating stress-enduring (Muse) cells in human dermal fibroblasts that are characterized by stress tolerance, expression of pluripotency markers, self-renewal, and the ability to differentiate into endodermal-, mesodermal-, and ectodermal-lineage cells from a single cell. They can be isolated as stage-specific embryonic antigen-3/CD105 double-positive cells. When human fibroblasts were separated into Muse and non-Muse cells and transduced with Oct3/4, Sox2, Klf4, and c-Myc, iPS cells were generated exclusively from Muse cells but not from non-Muse cells. Although some colonies were formed from non-Muse cells, they were unlike iPS cells. Furthermore, epigenetic alterations were not seen, and some of the major pluripotency markers were not expressed for the entire period during iPS cell generation. These findings were confirmed further using cells transduced with a single polycistronic virus vector encoding all four factors. The results demonstrate that in adult human fibroblasts a subset of preexisting adult stem cells whose properties are similar in some respects to those of iPS cells selectively become iPS cells, but the remaining cells make no contribution to the generation of iPS cells. Therefore this system seems to fit the elite model rather than the stochastic model.

Fluorescence polarization of green fluorescence protein

We report here the striking anisotropy of fluorescence exhibited by crystals of native green fluorescence protein (GFP). The crystals were generated by water dialysis of highly purified GFP obtained from the jellyfish Aequorea. We find that the fluorescence becomes six times brighter when the excitation, or emission, beam is polarized parallel (compared with perpendicular) to the crystal long axis. Thus, the major dipoles of the fluorophores must be oriented very nearly parallel to the crystal long axis. Observed in a polarizing microscope between parallel polars instead of either a polarizer or analyzer alone, the fluorescence polarization ratio rises to an unexpectedly high value of about 30:1, nearly the product of the fluorescence excitation and emission ratios, suggesting a sensitive method for measuring fluorophore orientations, even of a single fluorophore molecule. We have derived equations that accurately describe the relative fluorescence intensities of crystals oriented in various directions, with the polarizer and analyzer arranged in different configurations. The equations yield relative absorption and fluorescence coefficients for the four transition dipoles involved. Finally, we propose a model in which the elongated crystal is made of GFP molecules that are tilted 60° to align the fluorophores parallel to the crystal long axis. The unit layer in the model may well correspond to the arrangement of functional GFP molecules, to which resonant energy is efficiently transmitted from Ca2+-activated aequorin, in the jellyfish photophores.

Integumental reddish‐violet coloration owing to novel dichromatic chromatophores in the teleost fish, Pseudochromis diadema

In the reddish‐violet parts of the skin of the diadema pseudochromis Pseudochromis diadema, we found novel dichromatic chromatophores with a reddish pigment and reflecting platelets. We named these novel cells ‘erythro‐iridophores’. In standard physiological solution, erythro‐iridophores displayed two hues, red and dark violet when viewed with an optical microscope under ordinary transmission light and epi‐illumination optics, respectively. Under transmission electron microscopy, however, we observed no typical red chromatosomes, i.e., erythrosomes, in the cytoplasm. High‐performance thin‐layer chromatography (HPTLC) analysis of the pigment eluted from the erythro‐iridophores indicated that carotenoid is the main pigment generating the reddish color. Furthermore, when the irrigating medium was a K+‐rich saline solution, the color reflected from the erythro‐iridophores changed from dark violet to sky blue, but the red coloration remained. The motile activities of the erythro‐iridophores may participate in the changes in the reddish‐violet shades of the pseudochromis fish.

Centrifuge polarizing microscope. I. Rationale, design and instrument performance

We first describe early uses of the centrifuge for deciphering physical properties and molecular organization within living cells, as well as the development and use of centrifuge microscopes for such studies. The rationale for developing a centrifuge microscope that allows high‐extinction polarized light microscopy to observe dynamic fine structures in living cells is next discussed. We then describe a centrifuge polarizing microscope (CPM) that we developed for observing fine structural changes in living cells which are being exposed to up to ≈ 11 500 times earths gravitational field (g).

What is a vertebrate pigment cell

On the basis of discussions emerging from a workshop and discussions at the 7th meeting of the European Society for Pigment Cell Research in Geneva in 2012, this manuscript outlines useful criteria for defining the bona fide pigment cells as a functional entity of the vertebrate body plan and differentiating them from ‘pigmented’ cells in general. It also proposes a nomenclature for various types of pigment cells of vertebrates.

Novel Dichromatic Chromatophores in the Integument of the Mandarin Fish Synchiropus splendidus

The bluish coloration of the integument in teleosts is usually generated by light-reflecting chromatophores, called iridophores, that contain very thin light-reflecting platelets in the cytoplasm. We previously reported, however, that novel light-absorbing chromatophores named “cyanophores” display a bluish hue in the integument of the mandarin fish Synchiropus splendidus and the psychedelic fish S. picturatus. In the present report, we found novel light-absorbing dichromatic chromatophores in limited regions of the integument of the mandarin fish. Light microscopy under transmission optics revealed that the novel dichromatic chromatophores in the dermis around the margins of the bluish regions display both bluish and reddish hues. In the cytoplasm, both erythrosomes and cyanosomes were observed under transmission electron microscopy. The morphological features of the cyanosomes contained in the novel dichromatic chromatophores did not differ from those in the cyanophores in blue regions. Pharmacological treatment with norepinephrine induced aggregation of both the erythrosomes and cyanosomes of the novel dichromatic chromatophores, suggesting that the dichromatic chromatophores possess motile activity, the same as other lightabsorbing chromatophores, which contributes to the role of their color changes observed in response to various environmental signals. The variety of colors, patterns, and spectacular color changes the animals can exhibit is a subject of much interest. The generation of color and color changes is important to many species because it provides protection and assists in survival in their habitats. The delicate changes in hues and patterns can also be used to communicate with conspecifics.

Centrifuge polarizing microscope. II. Sample biological applications.

The rationale, design and general performance of the CPM (centrifuge polarizing microscope) were described in Part I of this study (Inouéet al. J. Microsc. 201 (2001) 341–356. In this second part, we describe observations on several biological samples that we have explored over the past two years using the CPM.

Calyculin A-induced neurite retraction is critically dependent on actomyosin activation but not on polymerization state of microtubules.

Calyculin A (CL-A), a toxin isolated from the marine sponge Discodermia calyx, is a strong inhibitor of protein phosphatase 1 (PP1) and 2A (PP2A). Although CL-A is known to induce rapid neurite retraction in developing neurons, the cytoskeletal dynamics of this retraction have remained unclear. Here, we investigated the cytoskeletal dynamics during CL-A-induced neurite retraction in cultured rat hippocampal neurons, using fluorescence microscopy as well as polarized light microscopy, which can visualize the polymerization state of the cytoskeleton in living cells. We observed that MTs were bent while maintaining their polymerization state during the neurite retraction. In addition, we also found that CL-A still induced neurite retraction when MTs were depolymerized by nocodazole or stabilized by paclitaxel. These results imply a mechanism other than depolymerization of MTs for CL-A-induced neurite retraction. Our pharmacological studies showed that blebbistatin and cytochalasin D, an inhibitor of myosin II and a depolymerizer of actin, strongly inhibited CL-A-induced neurite retraction. Based on all these findings, we propose that CL-A generates strong contractile forces by actomyosin to induce rapid neurite retraction independently from MT depolymerization.

Oocyte Maturation in Chaetopterus pergamentaceous Observed With Centrifuge Polarizing Microscope

Figure 1. Fluorescence localization of cytoskeletal proteins in cells trapped in