Tsutomu Motohashi

Generation of Structures Formed by Lens and Retinal Cells Differentiating From Embryonic Stem Cells

Embryonic stem cells have the potential to give rise to all cell lineages when introduced into the early embryo. They also give rise to a limited number of different cell types in vitro in specialized culture systems. In this study, we established a culture system in which a structure consisting of lens, neural retina, and pigmented retina was efficiently induced from embryonic stem cells. Refractile cell masses containing lens and neural retina were surrounded by retinal pigment epithelium layers and, thus, designated as eye‐like structures. Developmental processes required for eye development appear to proceed in this culture system, because the formation of the eye‐like structures depended on the expression of Pax6, a key transcription factor for eye development. The present culture system opens up the possibility of examining early stages of eye development and also of producing cells for use in cellular therapy for various diseases of the eye. Developmental Dynamics 228:664–671, 2003.

Multipotent cell fate of neural crest-like cells derived from embryonic stem cells.

Neural crest cells migrate throughout the embryo and differentiate into diverse derivatives: the peripheral neurons, cranial mesenchymal cells, and melanocytes. Because the neural crest cells have critical roles in organogenesis, detailed elucidation of neural crest cell differentiation is important in developmental biology. We recently reported that melanocytes could be induced from mouse ESCs. Here, we improved the culture system and showed the existence of neural crest‐like precursors. The addition of retinoic acid to the culture medium reduced the hematopoiesis and promoted the expression of the neural crest marker genes. The colonies formed contained neural crest cell derivatives: neurons and glial cells, together with melanocytes. This suggested that neural crest‐like cells assuming multiple cell fates had been generated in these present cultures. To isolate the neural crest‐like cells, we analyzed the expression of c‐Kit, a cell‐surface protein expressed in the early stage of neural crest cells in vivo. The c‐Kit‐positive (c‐Kit+) cells appeared as early as day 9 of the culture period and expressed the transcriptional factors Sox10 and Snail, which are expressed in neural crest cells. When the c‐Kit+ cells were separated from the cultures and recultured, they frequently formed colonies containing neurons, glial cells, and melanocytes. Even a single c‐Kit+ cell formed colonies that contained these three cell types, confirming their multipotential cell fate. The c‐Kit+ cells were also capable of migrating along neural crest migratory pathways in vivo. These results indicate that the c‐Kit+ cells isolated from melanocyte‐differentiating cultures of ESCs are closely related to neural crest cells.

Unexpected Multipotency of Melanoblasts Isolated from Murine Skin

Melanoblasts, precursor of melanocytes, are generated from the neural crest and differentiate into melanocytes during their migration throughout the entire body. The melanoblasts are thought to be progenitor cells that differentiate only into melanocyte. Here, we show that melanoblasts, even after they have already migrated throughout the skin, are multipotent, being able to generate neurons, glial cells, and smooth muscle cells in addition to melanocytes. We isolated Kit‐positive and CD45‐negative (Kit+/CD45−) cells from both embryonic and neonate skin by flow cytometry and cultured them on stromal cells. The Kit+/CD45− cells formed colonies containing neurons, glial cells, and smooth muscle cells, together with melanocytes. The Kit+/CD45− cells expressed Mitf‐M, Sox10, and Trp‐2, which are genes known to be expressed in melanoblasts. Even a single Kit+/CD45− cell formed colonies that contained neurons, glial cells, and melanocytes, confirming their multipotential cell fate. The colonies formed from Kit+/CD45− cells retained Kit+/CD45− cells even after 21 days in culture and these retained cells also differentiated into neurons, glial cells, and melanocytes, confirming their self‐renewal capability. When the Kit signal was inhibited by the antagonist ACK2, the Kit+/CD45− cells did not form colonies that contained multidifferentiated cells. These results indicate that melanoblasts isolated from skin have multipotency and self‐renewal capabilities. STEM CELLS 2009;27:888–897

Culture method for the induction of neurospheres from mouse embryonic stem cells by coculture with PA6 stromal cells

Embryonic stem (ES) cells proliferate and maintain their pluripotency for over 1 year in vitro and may therefore provide a sufficient source for cell therapies. However, most of the previously reported methods for obtaining a source for cell therapies have not been simple. We describe here a novel method for induction of neurospheres from mouse ES cells by coculturing on PA6 cells instead of the formation of embryoid bodies. The ES cells cocultured with the PA6 stromal cell line for at least 3 days were capable of differentiating into spheres. The cells in the spheres were all green fluorescent protein (GFP) positive, showing that they were derived from GFP‐expressing D3‐ES cells. The spheres contained nestin‐positive cells. The number of spheres increased when they were cocultured with PA6 for a longer period. Sphere formation was observed even after 10 mechanical dissociations and subculturings, showing its self‐renewal ability. The cells differentiated into microtubule‐associated protein‐2 (MAP2)‐positive neuronal cells and glial fibrillary acidic protein (GFAP)‐positive glial cells. γ‐Aminobutyric acid‐positive cells and tyrosine hydroxylase‐positive cells were also observed in the spheres. The percentages of the MAP2‐ or GFAP‐positive cells in the sphere changed according to the period of coculture on PA6 cells. At an early stage of coculture, more neurons were generated and, at a later period, more glial cells were generated. These results suggested that neurosphere could be generated from ES cells by coculturing with PA6, and that these cells resembled neural stem cells derived from mouse fetal brain tissue.

Cooperative and indispensable roles of endothelin 3 and KIT signalings in melanocyte development.

The development of melanocytes from neural crest‐derived precursor cells depends on signaling by the receptor tyrosine kinase KIT and the G protein‐coupled endothelin receptor B (EDNRB) pathways. Loss‐of‐function mutations in either of these two signaling receptor molecules cause a loss or a marked reduction in the number of melanocyte precursors in the embryo and finally lead to loss of the coat color. Using cultures of embryonic stem (ES) cells to induce melanocyte differentiation in vitro, we investigated the requirement for EDNRB signaling during the entire developmental process of the melanocyte, in association with that for KIT signaling. During the 21‐day period necessary for the induction of mature melanocytes from undifferentiated ES cells, endothelin 3 (EDN3), a ligand for EDNRB, increased the number of melanocytes in proportion to the period during which it was present. We tested the compensatory effect of EDNRB signaling on KIT signaling in vivo by using KitW‐LacZ/KitW‐LacZ ES cells and confirmed that the ectopic expression of EDN3 in the skin reduced the white spotting of KitW57/KitW57mice. KIT ligand (KITL) and EDN3 worked synergistically to induce melanocyte differentiation in vitro; however, the complete lack of EDNRB signaling attained by the use of EDN3−/− ES cells and an EDNRB antagonist, BQ788, revealed that the resulting failure of melanocyte development was not compensated by the further activation of KIT signaling by adding KITL. Simultaneous blockade of EDNRB and KIT signalings eliminated melanocyte precursors completely, suggesting that the maintenance or survival of early melanocyte precursors at least required the existence of either EDNRB or KIT signalings. Developmental Dynamics 233:407–417, 2005.

Maintenance of undifferentiated mouse embryonic stem cells in suspension by the serum- and feeder-free defined culture condition

The proven pluripotency of ES cells is expected to allow their therapeutic use for regenerative medicine. We present here a novel suspension culture method that facilitates the proliferation of pluripotent ES cells without feeder cells. The culture medium contains polyvinyl alcohol (PVA), free of either animal‐derived or synthetic serum, and contains very low amounts of peptidic or proteinaceous materials, which are favorable for therapeutic use. ES cells showed sustained proliferation in the suspension culture, and their undifferentiated state and pluripotency were experimentally verified. DNA microarray analyses showed a close relationship between the elevated expression of genes related to cell adhesions. We suggest that this suspension culture condition provides a better alternative to the conventional attached cell culture condition, especially for possible therapeutic use, by limiting the exposure of ES cells to feeder cells and animal products. Developmental Dynamics 237:2129–2138, 2008.

Tracing Sox10-expressing cells elucidates the dynamic development of the mouse inner ear.

The inner ear is constituted by complicated cochlear and vestibular compartments, which are derived from the otic vesicle, an embryonic structure of ectodermal origin. Although the inner ear development has been analyzed using various techniques, the developmental events have not been fully elucidated because of the intricate structure. We previously developed a Sox10-IRES-Venus mouse designed to express green fluorescent protein under the control of the Sox10 promoter. In the present study, we showed that the Sox10-IRES-Venus mouse enabled the non-destructive visualization and understanding of the morphogenesis during the development of the inner ear. The expression of the transcription factor Sox10 was first observed in the invaginating otic placodal epithelium, and continued to be expressed in the mature inner ear epithelium except for the hair cells and mesenchymal cells. We found that Sox10 was expressed in immature hair cells in the developing inner ear, suggesting that hair cells were generated from the Sox10-expressing prosensory cells. Furthermore, we demonstrated that scattered Sox10-expressing cells existed around the developing inner ear, some of which differentiated into pigmented melanocytes in the stria vascularis, suggesting that they were neural crest cells. Further analyzing the Sox10-IRES-Venus mice would provide important information to better understand the development of the inner ear.

Neural crest cells retain their capability for multipotential differentiation even after lineage-restricted stages.

Multipotency of neural crest cells (NC cells) is thought to be a transient phase at the early stage of their generation; after NC cells emerge from the neural tube, they are specified into the lineage‐restricted precursors. We analyzed the differentiation of early‐stage NC‐like cells derived from Sox10‐IRES‐Venus ES cells, where the expression of Sox10 can be visualized with a fluorescent protein. Unexpectedly, both the Sox10+/Kit− cells and the Sox10+/Kit+ cells, which were restricted in vivo to the neuron (N)‐glial cell (G) lineage and melanocyte (M) lineage, respectively, generated N, G, and M, showing that they retain multipotency. We generated mice from the Sox10‐IRES‐Venus ES cells and analyzed the differentiation of their NC cells. Both the Sox10+/Kit− cells and Sox10+/Kit+ cells isolated from these mice formed colonies containing N, G, and M, showing that they are also multipotent. These findings suggest that NC cells retain multipotency even after the initial lineage‐restricted stages. Developmental Dynamics 240:1681–1693, 2011.

The stemness of neural crest cells and their derivatives

Neural crest cells (NCCs) are unique to vertebrates and emerge from the border of the neural plate and subsequently migrate extensively throughout the embryo after which they differentiate into many types of cells. This multipotency is the main reason why NCCs are regarded as a versatile tool for stem cell biology and have been gathering attention for their potential use in stem cell based therapy. Multiple sets of networks comprised of signaling molecules and transcription factors regulate every developmental phase of NCCs, including maintenance of their multipotency. Pluripotent stem cell lines, such as embryonic stem cells and induced pluripotent stem (iPS) cells, facilitate the induction of NCCs in combination with sophisticated culture systems used for neural stem cells, although at present, clinical experiments for NCC-based cell therapy need to be improved. Unexpectedly, the multipotency of NCCs is maintained after they reach the target tissues as tissue neural crest stem cells (NCSCs) that may contribute to the establishment of NCC-derived multipotential stem cells. In addition, under specific culture conditions, fate-restricted unipotent descendants of NCCs, such as melanoblasts, show multipotency to differentiate into melanocytes, neurons, and glia cells. These properties contribute to the additional versatility of NCCs for therapeutic application and to better understand NCC development.

Protective Effect of Kit Signaling for Melanocyte Stem Cells against Radiation-Induced Genotoxic Stress

Radiation-induced hair graying is caused by irreversible defects in the self-renewal and/or development of follicular melanocyte stem cells in the hair follicles. Kit signaling is an essential growth and differentiation signaling pathway for various cell lineages including melanocytes, and its radioprotective effects have been shown in hematopoietic cells. However, it is uncertain whether Kit signaling exerts a radioprotective effect for melanocytes. In this study, we found that various loss-of-function mutations of Kit facilitate radiation-induced hair graying. In contrast, transgenic mice expressing the ligand for Kit (Kitl) in the epidermis have significantly reduced levels of radiation-induced hair graying. The X-ray doses used did not show a systemic lethal effect, indicating that the in vivo radiosensitivity of Kit mutants is mainly caused by the damaged melanocyte stem cell population. X-ray-damaged melanocyte stem cells seemed to take the fate of ectopically pigmented melanocytes in the bulge regions of hair follicles in vivo. Endothelin 3, another growth and differentiation factor for melanocytes, showed a lesser radioprotective effect compared with Kitl. These results indicate the prevention of radiation-induced hair graying by Kit signaling.