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ABSTRACT: In mice, coat pigmentation requires a stem cell (SC) system in which the survival, proliferation, and differentiation of melanocytes (MCs) are regulated by microenvironments in hair follicles (HFs). In vitro systems are required to analyze the behavior of single melanocyte stem cells (MCSCs) and their potential to form SC systems in vivo. We describe here an experimental system for the isolation, self-renewal, and differentiation of MCSCs, as well as an in vivo reconstitution assay for assessing their potential. Using Dct(tm1(Cre)Bee)/CAG-CAT-GFP mice, we show that, in the presence of stem cell factor and basic fibroblast growth factor and the XB2 feeder cell line, purified MCSCs can undergo clonogenic proliferation, resulting in c-Kit(low) side scatter(low) cells. In culture, these cells maintain their capacity to differentiate and reconstitute an MCSC system in HFs. As these cells are present in the upper part of the HF near the bulge region, express only low levels of housekeeping genes, and are resistant to neonatal treatment with ACK2, it is likely that only MCSCs that are quiescent in vivo have clonogenic activity in vitro. We also found that MCSCs can be purified from wild-type mice by fluorescent cell sorting and can be characterized in vitro.
Journal of Investigative Dermatology 07/2011; 131(12):2358-67. · 6.31 Impact Factor
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ABSTRACT: Hair follicle reconstitution analysis was used to test the contribution of melanocytes or their precursors to regenerated hair follicles. In this study, we first confirmed the process of chimeric hair follicle regeneration by both hair keratinocytes and follicular melanocytes. Then, as first suggested from the differential growth requirements of epidermal skin melanocytes and non-cutaneous or dermal melanocytes, we confirmed the inability of the latter to be involved as follicular melanocytes to regenerate hair follicles during the hair reconstitution assay. This clear functional discrimination between non-cutaneous or dermal melanocytes and epidermal melanocytes suggests the presence of two different melanocyte cell lineages, a finding that might be important in the pathogenesis of melanocyte-related diseases and melanomas.
Pigment Cell & Melanoma Research 11/2010; 24(1):125-35. · 5.06 Impact Factor
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Pigment Cell & Melanoma Research 10/2010; 23(5):683. · 5.06 Impact Factor
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ABSTRACT: Adult stem cells, which are characterized by their capacity for self-renewal and differentiation, participate in tissue homeostasis and response to injury. They are thought to enter a state of relative quiescence, known as reversible cell cycle arrest, but the underlying molecular mechanisms remain poorly characterized. Previous data from our laboratory has shown that housekeeping gene expression is downregulated in melanocyte stem cells (MelSCs), suggesting a global suppression of mRNA transcription. We now show, using antibodies against specific phosphorylated forms of RNA polymerase II (RNApII), that adult MelSCs do not undergo productive mRNA transcription elongation, while RNApII is activated and initialized, ready to synthesize mRNA upon stimulation, and that the RNApII kinase CDK9 is absent in adult MelSCs. Interestingly, other adult stem cells also, including keratinocyte, muscle, spermatogonia, and hematopoietic stem cells, showed a similar absence of RNApII phosphorylation. Although it is difficult to show the functional significance of this observation in vivo, CDK9 inhibition resulted in enhanced survival of cells that are deprived from survival factors. We conclude that the absence of productive mRNA transcription is an early, specific, and conserved characteristic of adult stem cells. Downregulation of mRNA transcription may lead to decreased rates of metabolism, and protection from cellular and genetic damage. Screening heterogeneous tissues, including tumors, for transcriptionally quiescent cells may result in the identification of cells with stem cell-like phenotypes.
Stem Cells 09/2010; 28(9):1571-80. · 7.78 Impact Factor
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ABSTRACT: Hematopoietic stem cell (HSC) proliferation is tightly regulated by a poorly understood complex of positive and negative cell-cycle regulatory mechanisms. Necdin (Ndn) is an evolutionally conserved multifunctional protein that has been implicated in cell-cycle regulation of neuronal cells. Here, we provide evidence that necdin plays an important role in restricting excessive HSC proliferation during hematopoietic regeneration. We identify Ndn as being preferentially expressed in the HSC population on the basis of gene expression profiling and demonstrate that mice deficient in Ndn show accelerated recovery of the hematopoietic system after myelosuppressive injury, whereas no overt abnormality is seen in steady-state hematopoiesis. In parallel, after myelosuppression, Ndn-deficient mice exhibit an enhanced number of proliferating HSCs. Based on these findings, we propose that necdin functions in a negative feedback loop that prevents excessive proliferation of HSCs during hematopoietic regeneration. These data suggest that the inhibition of necdin after clinical myelosuppressive treatment (eg, chemotherapy, HSC transplantation) may provide therapeutic benefits by accelerating hematologic recovery.
Blood 09/2009; 114(20):4383-92. · 9.90 Impact Factor
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ABSTRACT: The formation of functional skin entails multiple key signals that are implicated repeatedly in distinct processes during embryogenesis. Although Eph receptors and their membrane-bound ephrin ligands play a role in a wide variety of embryonic processes, their function in skin development has not been addressed. Here, we show that ephrin B2 is transiently expressed in hair buds during embryogenesis and in dermal mesenchymal cells during the perinatal period. Keratinocyte-specific ephrin B2-targeted mutant mice exhibit no skin phenotype, whereas postnatal systemic ephrin B2 ablation results in the enhancement of keratinocyte proliferation. Although the same treatment results in a defect of vascular remodeling, our analyses showed that the keratinocyte phenotype is not caused by hypoxia due to vascular defects. Interestingly, we found an enhanced expression of IL-1 family molecules, which have been implicated in the regulation of keratinocyte proliferation. On the basis of these observations, we propose that the transient expression of ephrin B2 in perinatal dermal mesenchymal cells plays a role in adjusting the activity of the mesenchymal microenvironment that regulates proliferation of keratinocytes.
Journal of Investigative Dermatology 08/2009; 129(10):2386-95. · 6.31 Impact Factor
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ABSTRACT: Notch signaling is an evolutionally conserved pathway that serves as a critical regulator of cell fate. From a series of studies, including a report in this issue, researchers have begun to elucidate the critical functions of Notch signaling in the regulation of melanocyte lineage development. With evidence of a recently identified role for Notch signaling in melanomagenesis, characterization of downstream molecular events may offer potential avenues for the development of novel therapeutic strategies.
Journal of Investigative Dermatology 12/2008; 128(11):2571-4. · 6.31 Impact Factor
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Tae Inoue-Narita,
Koichi Hamada,
Takehiko Sasaki,
Sachiko Hatakeyama,
Sachiko Fujita,
Kohichi Kawahara,
Masato Sasaki,
Hiroyuki Kishimoto,
Satoshi Eguchi,
Itaru Kojima,
Friedrich Beermann,
Tetsunori Kimura, Masatake Osawa,
Satoshi Itami,
Tak Wah Mak,
Toru Nakano,
Motomu Manabe,
Akira Suzuki
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ABSTRACT: Phosphate and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor gene inactivated in numerous sporadic cancers, including melanomas. To analyze Pten functions in melanocytes, we used the Cre-loxP system to delete Pten specifically in murine pigment-producing cells and generated DctCrePten(flox/flox) mice. Half of DctCrePten(flox/flox) mice died shortly after birth with enlargements of the cerebral cortex and hippocampus. Melanocytes were increased in the dermis of perinatal DctCrePten(flox/flox) mice. When the mutants were subjected to repeated depilations, melanocyte stem cells in the bulge of the hair follicle resisted exhaustion and the mice were protected against hair graying. Although spontaneous melanomas did not form in DctCrePten(flox/flox) mice, large nevi and melanomas developed after carcinogen exposure. DctCrePten(flox/flox) melanocytes were increased in size and exhibited heightened activation of Akt and extracellular signal-regulated kinases, increased expression of Bcl-2, and decreased expression of p27(Kip1). Our results show that Pten is important for the maintenance of melanocyte stem cells and the suppression of melanomagenesis.
Cancer Research 07/2008; 68(14):5760-8. · 7.86 Impact Factor
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ABSTRACT: Recent studies have shown that Notch signaling plays an important role in epidermal development, but the underlying molecular mechanisms remain unclear. Here, by integrating loss- and gain-of-function studies of Notch receptors and Hes1, we describe molecular information about the role of Notch signaling in epidermal development. We show that Notch signaling determines spinous cell fate and induces terminal differentiation by a mechanism independent of Hes1, but Hes1 is required for maintenance of the immature state of spinous cells. Notch signaling induces Ascl2 expression to promote terminal differentiation, while simultaneously repressing Ascl2 through Hes1 to inhibit premature terminal differentiation. Despite the critical role of Hes1 in epidermal development, Hes1 null epidermis transplanted to adult mice showed no obvious defects, suggesting that this role of Hes1 may be restricted to developmental stages. Overall, we conclude that Notch signaling orchestrates the balance between differentiation and immature programs in suprabasal cells during epidermal development.
Developmental cell 05/2008; 14(4):594-604. · 13.36 Impact Factor
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ABSTRACT: Elucidation of molecular mechanisms underlying stem cell regulation is of great importance for their clinical applications
in regenerative medicine and cancer therapy. The function of stem cells is maintained by their specialized microenvironment,
referred as the niche. Despite intensive studies of the stem cell niche, the molecular basis of stem cell regulation by the
niche has still remained elusive. Since molecular interactions between stem cells and the niche can be analyzed only under
in vivo conditions, one drawback that hampers stem cell research is the lack of an efficient in vivo assay system that allow to define an in vivo gene function for the regulation of stem cells. We have previously identified melanocyte stem cells (MSCs) in the mouse hair
follicle, in which MSCs reside at a specific region of the hair follicle, termed as the lower permanent portion. MSCs offer
an attractive model with which to study the molecular basis of stem cell regulation, because loss-of-function mutations in
the genes responsible for MSC regulation are readily identifiable by a premature hair graying phenotype in mice. This implies
the irresistible possibility that MSCs allows us to identify the genes involved in stem cell regulation by a phenotype-driven
genetic screen in mice. Hence, we believe that MSC system is an excellent model to study stem cell biology.
03/2008: pages 129-144;
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ABSTRACT: Elucidation of the molecular mechanisms underlying stem cell regulation is of great importance both for basic biology and for clinical applications. Melanocyte stem cells (MSCs) are an excellent model in which to study the molecular basis of stem cell regulation, as the genetic alterations involved in the maintenance of the stem cells are readily identifiable by a premature hair graying phenotype. Research on MSCs has been hampered by the lack of a reliable system to assay their function. Here, by co-culturing highly purified melanoblasts (MBs) with XB2 keratinocytes, we describe an efficient culture method that allows the expansion of immature MBs in vitro. These MBs are also capable of undergoing terminal differentiation into mature melanocytes (MCs) when differentiation is induced. Furthermore, by performing a hair-follicle reconstitution assay in which expanded MBs in a mixture of epidermal and dermal cells were grafted to reconstitute a hair follicle, we demonstrate that the expanded MBs retain their capacity to become incorporated into newly developed hair follicles and repopulate the MC stem cell population there. Thus, by integrating genetic manipulations in cultured MBs in vitro, this method provides a powerful tool with which to study the molecular basis of stem cell regulation.
Journal of Investigative Dermatology 03/2008; 128(2):408-20. · 6.31 Impact Factor
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ABSTRACT: As the ability to detect and define stem cells (SCs) has increased, attention is turning towards the definition of the niche and the identification of the signals that induce SC quiescence or proliferation. The melanocyte stem-niche system, in which the SCs and their progeny occupy geographically distinct domains within the hair follicle, provides one of the best models for studying the complex interplay between environmental cues and transcription factors that underpin cell fate. This review discusses what is known of the origin and molecular characteristics of melanocyte SCs and proposes a series of temporal events that are likely to contribute to the establishment of melanocyte SCs in the hair follicle. We also highlight the possibility of in vitro systems capable of directing cultured melanocytes/melanoblasts to a SC fate in response to specific extrinsiccues.
Pigment Cell Research 09/2007; 20(4):263-70. · 4.29 Impact Factor
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Mariko Moriyama, Masatake Osawa,
Siu-Shan Mak,
Toshiyuki Ohtsuka,
Norio Yamamoto,
Hua Han,
Véronique Delmas,
Ryoichiro Kageyama,
Friedrich Beermann,
Lionel Larue,
Shin-Ichi Nishikawa
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ABSTRACT: Melanoblasts (Mbs) are thought to be strictly regulated by cell-cell interactions with epidermal keratinocytes, although the precise molecular mechanism of the regulation has been elusive. Notch signaling, whose activation is mediated by cell-cell interactions, is implicated in a broad range of developmental processes. We demonstrate the vital role of Notch signaling in the maintenance of Mbs, as well as melanocyte stem cells (MSCs). Conditional ablation of Notch signaling in the melanocyte lineage leads to a severe defect in hair pigmentation, followed by intensive hair graying. The defect is caused by a dramatic elimination of Mbs and MSCs. Furthermore, targeted overexpression of Hes1 is sufficient to protect Mbs from the elimination by apoptosis. Thus, these data provide evidence that Notch signaling, acting through Hes1, plays a crucial role in the survival of immature Mbs by preventing initiation of apoptosis.
The Journal of Cell Biology 06/2006; 173(3):333-9. · 10.26 Impact Factor
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ABSTRACT: Bcl2 null mice display a characteristic loss of pigmentation demonstrating the importance of Bcl2 in the melanocyte (Mc) lineage. It was recently reported that this abnormal phenotype is due to the failure of melanocyte stem cell (MSC) maintenance and that Bcl2 is selectively important for the survival of MSCs. However, in our analysis of the same mouse, we observe a reduction in melanoblast (Mb) number in both epidermal and follicular populations. More importantly, there is a complete absence of MSCs. SCF downregulation in the epidermis is concomitant with the dramatic reduction in Mb numbers observed in the Bcl2 null, suggesting that Bcl2 is indispensable for the survival of Mbs in the absence of c-Kit signaling. Consistently, abrogation of c-Kit signaling in Bcl2 null mice depletes all Mbs and Mcs, whereas continuous expression of SCF in epidermal keratinocytes rescues the MSCs. Our results demonstrate that Bcl2 has a general role in Mb and Mc survival and is essential for the emergence of MSCs. Moreover, the results indicate that the first wave of Mcs that provide hair pigmentation is derived directly from epidermal Mbs bypassing MSCs. Furthermore, a Bcl2-independent mechanism of action of SCF in the Mc lineage is revealed as SCF c-Kit signaling is functional in the absence of Bcl2.
Developmental Biology 04/2006; 291(1):144-53. · 4.07 Impact Factor
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ABSTRACT: Emerging evidence from stem cell (SC) research has strengthened the idea that SC fate is determined by a specialized environment, known as the SC niche. However, because of the difficulty of identifying individual stem cells and their surrounding components in situ, the exact mechanisms underlying SC regulation by the niche remain elusive. To overcome this difficulty, we employed melanocyte stem cells (MSCs), which allow the identification of individual SCs in the niche, the lower permanent portion of the hair follicle (HF). Here, we present molecular makers that can distinguish MSCs from other melanocyte (MC) subsets in the HF. We also describe a simple and robust method that allows gene expression profiling in individual SCs. After isolating individual MSCs from transgenic mice in which the MCs are marked by green fluorescence protein (GFP), we performed single-cell transcript analysis to obtain the molecular signature of individual MSCs in the niche. The data suggest the existence of a mechanism that induces the downregulation of various key molecules for MC proliferation or differentiation in MSCs located in the niche. By integrating these data, we propose that the niche is an environment that insulates SCs from various activating stimuli and maintains them in a quiescent state.
Development 01/2006; 132(24):5589-99. · 6.60 Impact Factor
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ABSTRACT: Mouse epidermal melanoblasts and melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanocyte-proliferation medium (MDMD) and a melanoblast-proliferation medium (MDMDF) supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). Pure cultured primary melanoblasts and melanocytes were further cultured with MDMD/MDMDF supplemented with granulocyte-macrophage colony-stimulating factor (GMCSF) from 14 days (keratinocyte depletion). GMCSF stimulated the number of melanoblasts/melanocytes as well as the percentage of differentiated melanocytes in keratinocyte-depleted cultures. Flow cytometry analysis showed that melanoblasts and melanocytes in the S and G(2)/M phases of the cell cycle were increased by the treatment with GMCSF. Moreover, anti-GMCSF antibody added to MDMD/MDMDF from the initiation of the primary culture (in the presence of keratinocytes) inhibited the proliferation of melanoblasts/melanocytes as well as the differentiation of melanocytes. Enzyme-linked immunosorbent assay of culture media revealed that GMCSF was secreted from keratinocytes, but not from melanocytes. These results suggest that GMCSF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanoblasts/melanocytes in culture in cooperation with cAMP elevator and bFGF.
Experimental Cell Research 08/2004; 297(2):593-606. · 3.58 Impact Factor
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Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 06/2004; 49(6):727-33.
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ABSTRACT: Mouse epidermal melanoblasts and melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanocyte-proliferation medium (MDMD) and melanoblast-proliferation medium (MDMDF) supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). Pure cultured primary melanoblasts and melanocytes were further cultured with MDMD/MDMDF supplemented with hepatocyte growth factor (HGF) from 14 days (keratinocyte depletion). The HGF increased the number of melanoblasts and melanocytes, but not the percentage of differentiated melanocytes in the melanoblast-melanocyte population in the absence of keratinocytes. Flow cytometry analysis showed that melanoblasts and melanocytes in the S and/or G2/M phases of the cell cycle were increased by the treatment with HGF. Moreover, an anti-HGF antibody supplemented to MDMD/MDMDF from the initiation of the primary culture (in the presence of keratinocytes) inhibited the proliferation of melanoblasts and melanocytes, but not the differentiation of melanocytes. These results suggest that HGF is a keratinocyte-derived factor involved in regulating the proliferation of epidermal melanoblasts and melanocytes from newborn mice in cooperation with cAMP elevators and/or bFGF.
Pigment Cell Research 03/2004; 17(1):51-61. · 4.29 Impact Factor
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ABSTRACT: Mouse epidermal melanoblasts and melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanocyte-proliferation medium (MDMD) and melanoblast-proliferation medium (MDMDF) supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). Pure cultured primary melanoblasts and melanocytes were then further cultured with MDMD/MDMDF supplemented with steel factor (SLF) (keratinocyte depletion). SLF increased the number of melanoblasts and melanocytes as well as the proportion of differentiated melanocytes in the absence of keratinocytes. Flow cytometric analysis showed that melanoblasts and melanocytes in the S and G2/M phases of the cell cycle were increased by treatment with SLF. Moreover, an anti-SLF antibody added to MDMD/MDMDF from the initiation of the primary culture (in the presence of keratinocytes) inhibited the proliferation of melanoblasts and melanocytes as well as the differentiation of melanocytes. These results suggest that SLF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture in cooperation with cAMP elevator and bFGF.
Pigment Cell Research 01/2004; 16(6):644-55. · 4.29 Impact Factor
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Atsushi Kunisato,
Shigeru Chiba,
Etsuko Nakagami-Yamaguchi,
Keiki Kumano,
Toshiki Saito,
Shigeo Masuda,
Tomoyuki Yamaguchi, Masatake Osawa,
Ryoichiro Kageyama,
Hiromitsu Nakauchi,
Mitsuo Nishikawa,
Hisamaru Hirai
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ABSTRACT: Mouse long-term hematopoietic reconstituting cells exist in the c-Kit+Sca-1+Lin- (KSL) cell population; among them, CD34(low/-) cells represent the most highly purified population of hematopoietic stem cells in the adult bone marrow. Here, we demonstrate that retrovirus-mediated transduction of CD34(low/-)c-Kit+Sca-1+Lin- (34-KSL) cells with the HES-1 gene, which encodes a basic helix-loop-helix transcription factor functioning downstream of the Notch receptor, and is a key molecule for the growth phase of neural stem cells in the embryo, preserves the long-term reconstituting activity of these cells in vitro. We also show that cells derived from the HES-1-transduced 34-KSL population produce progenies characterized by negative Hoechst dye staining, which defines the side population, and by CD34(low/-) profile in the bone marrow KSL population in each recipient mouse at ratios 3.5- and 7.8-fold those produced by nontransduced 34-KSL-derived competitor cells. We conclude that HES-1 preserves the long-term reconstituting hematopoietic activity of 34-KSL stem cells ex vivo. Up-regulation of HES-1 protein in the 34-KSL population before unnecessary cell division, that is, without retrovirus transduction, may represent a potent approach to absolute expansion of hematopoietic stem cells.
Blood 04/2003; 101(5):1777-83. · 9.90 Impact Factor
