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Harufumi Yamasaki, Aiko Sada, Takeyuki Iwata, Tohru Niwa, Minoru Tomizawa, Kleanthis G. Xanthopoulos, Toru Koike, Nobuyoshi Shiojiri, Suppression of C/EBP alpha expression in periportal hepatoblasts may stimulate biliary cell differentiation through increased Hnf6 and Hnf1b expression, DEVELOPMENT, 10.1242/dev.02591, 133, 21, 4233-4243, 2006.11, The expression of C/EBP alpha, which may govern transcription of mature hepatocyte marker genes, was suppressed in periportal hepatoblasts in mouse liver development, leading to biliary cell differentiation. This study was undertaken to analyze how inactivation of the Cebpa gene affects biliary cell differentiation and gene expression of the regulatory genes for that differentiation, including Hnf1b and Hnf6. In the knockout mouse liver at midgestation stages, pseudoglandular structures were abundantly induced in the parenchyma with elevated expression of Hnf6 and Hnf1b mRNAs. The wild-type liver parenchyma expressed mRNAs of these transcription factors at low levels, though periportal biliary progenitors had strong expression of them. These results suggest that expression of Hnf6 and Hnf1b is downstream of C/EBP alpha action in fetal liver development, and that the suppression of C/EBP alpha expression in periportal hepatoblasts may lead to expression of Hnf6 and Hnf1b mRNAs. Immunohistochemical studies with biliary cell markers in knockout livers demonstrated that differentiated biliary epithelial cells were confined to around the portal veins. The suppression of C/EBP alpha expression may result in upregulation of Hnf6 and Hnf1b gene expression, but be insufficient for biliary cell differentiation. When liver fragments of Cebpa-knockout fetuses, in which hepatoblasts were contained as an endodermal component, were transplanted in the testis of Scid (Prkdc) male mice, almost all hepatoblasts gave rise to biliary epithelial cells. Wild-type hepatoblasts constructed mature hepatic tissue accompanied by biliary cell differentiation. These results also demonstrate that the suppression of C/EBP alpha expression may stimulate biliary cell differentiation..  |
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Hitomi Suzuki, Aiko Sada, Shosei Yoshida, Yumiko Saga, The heterogeneity of spermatogonia is revealed by their topology and expression of marker proteins including the germ cell-specific proteins Nanos2 and Nanos3, DEVELOPMENTAL BIOLOGY, 10.1016/j.ydbio.2009.10.002, 336, 2, 222-231, 2009.12, Spermatogonial stem cells (SSCs) reside in undifferentiated type-A spermatogonia and contribute to continuous spermatogenesis by maintaining the balance between self-renewal and differentiation, thereby meeting the biological demand in the testis. Spermatogonia have to date been characterized principally through their morphology, but we herein report the detailed characterization of undifferentiated spermatogonia in mouse testes based on their gene expression profiles in combination with topological features. The detection of the germ cell-specific proteins Nanos2 and Nanos3 as markets of spermatogonia has enabled the clear dissection of complex populations of these cells as Nanos2 was recently shown to be involved in the maintenance of stem cells. Nanos2 is found to be almost exclusively expressed in A(s) to A(pr) cells, whereas Nanos3 is detectable in most undifferentiated spermatogonia (A(s) to A(al)) and differentiating A(1) spermatogonia. In our present study, we find that A(s) and A(pr) can be basically classified into three categories: (1) GFR alpha 1(+)Nanos2(+)Nanos3(-)Ngn3(-), (2) GFR alpha 1(+)Nanos2(+)Nanos3(+)Ngn3(-), and (3) GFR alpha 1(-)Nanos2(+/-)Nanos3(+)Ngn3(+). We propose that the first of these groups is most likely to include the stem cell population and that Nanos3 may function in transit amplifying cells. (C) 2009 Elsevier Inc. All rights reserved.. |
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Aiko Sada, Atsushi Suzuki, Hitomi Suzuki, Yumiko Saga, The RNA-Binding Protein NANOS2 Is Required to Maintain Murine Spermatogonial Stem Cells, SCIENCE, 10.1126/science.1172645, 325, 5946, 1394-1398, 2009.09, Stem cells give rise to differentiated cell types but also preserve their undifferentiated state through cell self-renewal. With the use of transgenic mice, we found that the RNA-binding protein NANOS2 is essential for maintaining spermatogonial stem cells. Lineage-tracing analyses revealed that undifferentiated spermatogonia expressing Nanos2 self-renew and generate the entire spermatogenic cell lineage. Conditional disruption of postnatal Nanos2 depleted spermatogonial stem cell reserves, whereas mouse testes in which Nanos2 had been overexpressed accumulated spermatogonia with undifferentiated, stem cell-like properties. Thus, NANOS2 is a key stem cell regulator that is expressed in self-renewing spermatogonial stem cells and maintains the stem cell state during murine spermatogenesis.. |
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Aiko Sada, Kazuteru Hasegawa, Pui Han Pin, Yumiko Saga, NANOS2 Acts Downstream of Glial Cell Line-Derived Neurotrophic Factor Signaling to Suppress Differentiation of Spermatogonial Stem Cells, STEM CELLS, 10.1002/stem.790, 30, 2, 280-291, 2012.02, Stem cells are maintained by both stem cell-extrinsic niche signals and stem cell-intrinsic factors. During murine spermatogenesis, glial cell line-derived neurotrophic factor (GDNF) signal emanated from Sertoli cells and germ cell-intrinsic factor NANOS2 represent key regulators for the maintenance of spermatogonial stem cells. However, it remains unclear how these factors intersect in stem cells to control their cellular state. Here, we show that GDNF signaling is essential to maintain NANOS2 expression, and overexpression of Nanos2 can alleviate the stem cell loss phenotype caused by the depletion of Gfra1, a receptor for GDNF. By using an inducible Cre-loxP system, we show that NANOS2 expression is downregulated upon the conditional knockout (cKO) of Gfra1, while ectopic expression of Nanos2 in GFRA1-negative spermatogonia does not induce de novo GFRA1 expression. Furthermore, overexpression of Nanos2 in the Gfra1-cKO testes prevents precocious differentiation of the Gfra1-knockout stem cells and partially rescues the stem cell loss phenotypes of Gfra1-deficient mice, indicating that the stem cell differentiation can be suppressed by NANOS2 even in the absence of GDNF signaling. Taken together, we suggest that NANOS2 acts downstream of GDNF signaling to maintain undifferentiated state of spermatogonial stem cells. STEM CELLS 2012; 30:280291.. |
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Song Eun Lee, Aiko Sada, Meng Zhang, David J. McDermitt, Shu Yang Lu, Kenneth J. Kemphues, Tudorita Tumbar, High Runx1 Levels Promote a Reversible, More-Differentiated Cell State in Hair-Follicle Stem Cells during Quiescence, CELL REPORTS, 10.1016/j.celrep.2013.12.039, 6, 3, 499-513, 2014.02, Quiescent hair follicle (HF) bulge stem cells (SCs) differentiate to early progenitor (EP) hair germ (HG) cells, which divide to produce transit-amplifying matrix cells. EPs can revert to SCs upon injury, but whether this dedifferentiation occurs in normal HF homeostasis (hair cycle) and the mechanisms regulating both differentiation and dedifferentiation are unclear. Here, we use lineage tracing, gain of function, transcriptional profiling, and functional assays to examine the role of observed endogenous Runx1 level changes in the hair cycle. We find that forced Runx1 expression induces hair degeneration (catagen) and simultaneously promotes changes in the quiescent bulge SC transcriptome toward a cell state resembling the EP HG fate. This cell-state transition is functionally reversible. We propose that SC differentiation and dedifferentiation are likely to occur during normal HF degeneration and niche restructuring in response to changes in endogenous Runx1 levels associated with SC location with respect to the niche.. |
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Zhi Zhou, Takayuki Shirakawa, Kazuyuki Ohbo, Aiko Sada, Quan Wu, Kazuteru Hasegawa, Rie Saba, Yumiko Saga, RNA Binding Protein Nanos2 Organizes Post-transcriptional Buffering System to Retain Primitive State of Mouse Spermatogonial Stem Cells, DEVELOPMENTAL CELL, 10.1016/j.devcel.2015.05.014, 34, 1, 96-107, 2015.07, In many adult tissues, homeostasis relies on self-renewing stem cells that are primed for differentiation. The reconciliation mechanisms of these characteristics remain a fundamental question in stem cell biology. We propose that regulation at the post-transcriptional level is essential for homeostasis in murine spermatogonial stem cells (SSCs). Here, we show that Nanos2, an evolutionarily conserved RNA-binding protein, works with other cellular messenger ribonucleoprotein (mRNP) components to ensure the primitive status of SSCs through a dual mechanism that involves (1) direct recruitment and translational repression of genes that promote spermatogonial differentiation and (2) repression of the target of rapamycin complex 1 (mTORC1), a well-known negative pathway for SSC self-renewal, by sequestration of the core factor mTOR in mRNPs. This mechanism links mRNA turnover to mTORC1 signaling through Nanos2-containing mRNPs and establishes a post-transcriptional buffering system to facilitate SSC homeostasis in the fluctuating environment within the seminiferous tubule.. |
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Aiko Sada, Fadi Jacob, Eva Leung, Sherry Wang, Brian S. White, David Shalloway, Tudorita Tumbar, Defining the cellular lineage hierarchy in the interfollicular epidermis of adult skin, NATURE CELL BIOLOGY, 10.1038/ncb3359, 18, 6, 619-+, 2016.06, The interfollicular epidermis regenerates from heterogeneous basal skin cell populations that divide at different rates. It has previously been presumed that infrequently dividing basal cells known as label-retaining cells (LRCs) are stem cells, whereas non-LRCs are short-lived progenitors. Here we employ the H2B-GFP pulse-chase system in adult mouse skin and find that epidermal LRCs and non-LRCs are molecularly distinct and can be differentiated by Dlx1(CreER) and Slc1a3(CreER) genetic marking, respectively. Long-term lineage tracing and mathematical modelling of H2B-GFP dilution data show that LRCs and non-LRCs constitute two distinct stem cell populations with different patterns of proliferation, differentiation and upward cellular transport. During homeostasis, these populations are enriched in spatially distinct skin territories and can preferentially produce unique differentiated lineages. On wounding or selective killing, they can temporarily replenish each other's territory. These two discrete interfollicular stem cell populations are functionally interchangeable and intrinsically well adapted to thrive in distinct skin environments.. |
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Aiko Sada, Prachi Jain, Sherry Wang, Eva Leung, Tudorita Tumbar, Slc1a3-CreER as a Targeting Tool for the K6+ Epithelial Stem Cell Niche and its Precursors during Mouse Hair Follicle Cycle, Journal of Investigative Dermatology, 10.1016/j.jid.2017.02.974, 137, 7, 1569-1571, 2017.07. |
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Changarathil G, Ramirez K, Isoda H, Sada A, Yanagisawa H, Wild-type and SAMP8 mice show age-dependent changes in distinct stem cell compartments of the interfollicular epidermis, PLoS One, 10.1371/journal.pone.0215908, 14, 5, e0215908, 2019.05. |
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Kang S, Long K, Wang S, Sada A, Tumbar T, Histone H3 K4/9/27 trimethylation levels affect wound healing and stem cell dynamics in adult skin, Stem Cell Reports, 10.1016/j.stemcr.2019.11.007, 14, 1, 34-48, 2020.01. |
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Lalhaba Oinam, Gopakumar Changarathil, Erna Raja, Yen Xuan Ngo, Hiroaki Tateno, Aiko Sada, Hiromi Yanagisawa, Glycome profiling by lectin microarray reveals dynamic glycan alterations during epidermal stem cell aging, Aging Cell, 10.1111/acel.13190, 19, 8, e13190, 2020.07, Aging in the epidermis is marked by a gradual decline in barrier function, impaired wound healing, hair loss, and an increased risk of cancer. This could be due to age-related changes in the properties of epidermal stem cells and defective interactions with their microenvironment. Currently, no biochemical tools are available to detect and evaluate the aging of epidermal stem cells. The cellular glycosylation is involved in cell-cell communications and cell-matrix adhesions in various physiological and pathological conditions. Here, we explored the changes of glycans in epidermal stem cells as a potential biomarker of aging. Using lectin microarray, we performed a comprehensive glycan profiling of freshly isolated epidermal stem cells from young and old mouse skin. Epidermal stem cells exhibited a significant difference in glycan profiles between young and old mice. In particular, the binding of a mannose-binder rHeltuba was decreased in old epidermal stem cells, whereas that of an α2-3Sia-binder rGal8N increased. These glycan changes were accompanied by upregulation of sialyltransferase, St3gal2 and St6gal1 and mannosidase Man1a genes in old epidermal stem cells. The modification of cell surface glycans by overexpressing these glycogenes leads to a defect in the regenerative ability of epidermal stem cells in culture. Hence, our study suggests the age-related global alterations in cellular glycosylation patterns and its potential contribution to the stem cell function. These glycan modifications detected by lectins may serve as molecular markers for aging, and further functional studies will lead us to a better understanding of the process of skin aging.. |
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Ryutaro Ishii, Hiromi Yanagisawa, Aiko Sada, Defining compartmentalized stem cell populations with distinct cell division dynamics in the ocular surface epithelium, Development, 10.1242/dev.197590, 147, 24, dev197590-dev197590, 2020.12, ABSTRACTAdult tissues contain label-retaining cells (LRCs), which are relatively slow-cycling and considered to represent a property of tissue stem cells (SCs). In the ocular surface epithelium, LRCs are present in the limbus and conjunctival fornix; however, the character of these LRCs remains unclear, owing to lack of appropriate molecular markers. Using three CreER transgenic mouse lines, we demonstrate that the ocular surface epithelium accommodates spatially distinct populations with different cell division dynamics. In the limbus, long-lived Slc1a3CreER-labeled SCs either migrate centripetally toward the central cornea or slowly expand their clones laterally within the limbal region. In the central cornea, non-LRCs labeled with Dlx1CreER and K14CreER behave as short-lived progenitor cells. The conjunctival epithelium in the bulbar, fornix and palpebral compartment is regenerated by regionally unique SC populations. Severe damage to the cornea leads to the cancellation of SC compartments and conjunctivalization, whereas milder limbal injury induces a rapid increase of laterally expanding clones in the limbus. Taken together, our work defines compartmentalized multiple SC/progenitor populations of the mouse eye in homeostasis and their behavioral changes in response to injury.. |
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Yen Xuan Ngo, Kenta Haga, Ayako Suzuki, Hiroko Kato, Hiromi Yanagisawa, Kenji Izumi, Aiko Sada, Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate., Journal of visualized experiments : JoVE, 10.3791/62820, 175, 2021.09, For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and α6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFRα. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.. |
