Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells.

Hematopoietic stem cells (HSCs) undergo self-renewing cell divisions and maintain blood production for their lifetime. Appropriate control of HSC self-renewal is crucial for the maintenance of hematopoietic homeostasis. Here we show that activation of p38 MAPK in response to increasing levels of reactive oxygen species (ROS) limits the lifespan of HSCs in vivo. In Atm(-/-) mice, elevation of ROS levels induces HSC-specific phosphorylation of p38 MAPK accompanied by a defect in the maintenance of HSC quiescence. Inhibition of p38 MAPK rescued ROS-induced defects in HSC repopulating capacity and in the maintenance of HSC quiescence, indicating that the ROS-p38 MAPK pathway contributes to exhaustion of the stem cell population. Furthermore, prolonged treatment with an antioxidant or an inhibitor of p38 MAPK extended the lifespan of HSCs from wild-type mice in serial transplantation experiments. These data show that inactivation of p38 MAPK protects HSCs against loss of self-renewal capacity. Our characterization of molecular mechanisms that limit HSC lifespan may lead to beneficial therapies for human disease.

Identification of tumor-initiating cells in a highly aggressive brain tumor using promoter activity of nucleostemin.

Controversy remains over whether the cancer stem cell (CSC) theory applies to all tumors. To determine whether cells within a highly aggressive solid tumor are stochastically or hierarchically organized, we combined a reporter system where the nucleostemin (NS) promoter drives GFP expression (termed NS-GFP) with a mouse brain tumor model induced by retroviral Ras expression on a p16(Ink4a)/p19(Arf)-deficient background. The NS-GFP system allowed us to monitor the differentiation process of normal neural stem/precursor cells by analyzing GFP fluorescence intensity. In tumor-bearing mice, despite the very high frequency of tumorigenic cells, we successfully identified the NS-GFP(+) cells as tumor-initiating cells (T-ICs). The clonal studies conclusively established that phenotypical heterogeneity can exist among the cells comprising a genetically homogeneous tumor, suggesting that this aggressive brain tumor follows the CSC model. Detailed analyses of the NS-GFP(+) brain tumor cells revealed that T-ICs showed activation of the receptor tyrosine kinase c-Met, which functions in tumor invasiveness. Thus, the NS-GFP system provides a powerful tool to elucidate stem cell biology in normal and malignant tissues.

Identification of stem cells during prepubertal spermatogenesis via monitoring of nucleostemin promoter activity.

The nucleostemin (NS) gene encodes a nucleolar protein found at high levels in several types of stem cells and tumor cell lines. The function of NS is unclear but it may play a critical role in S-phase entry by stem/progenitor cells. Here we characterize NS expression in murine male germ cells. Although NS protein was highly expressed in the nucleoli of all primordial germ cells, only a limited number of gonocytes showed NS expression in neonatal testes. In adult testes, NS protein was expressed at high levels in the nucleoli of spermatogonia and primary spermatocytes but at only low levels in round spermatids. To evaluate the properties of cells expressing high levels of NS, we generated transgenic reporter mice expressing green fluorescent protein (GFP) under the control of the NS promoter (NS-GFP Tg mice). In adult NS-GFP Tg testes, GFP and endogenous NS protein expression were correlated in spermatogonia and spermatocytes but GFP was also ectopically expressed in elongated spermatids and sperm. In testes of NS-GFP Tg embryos, neonates, and 10-day-old pups, however, GFP expression closely coincided with endogenous NS expression in developing germ cells. In contrast to a previous report, our results support the existence in neonatal testes of spermatogonial stem cells with long-term repopulating capacity. Furthermore, our data show that NS expression does not correlate with cell-cycle status during prepuberty, and that strong NS expression is essential for the maintenance of germline stem cell proliferation capacity. We conclude that NS is a marker of undifferentiated status in the germ cell lineage during prepubertal spermatogenesis.

A CTX family cell adhesion molecule, JAM4, is expressed in stem cell and progenitor cell populations of both male germ cell and hematopoietic cell lineages.

Stem cells are maintained in an undifferentiated state by interacting with a microenvironment known as the "niche," which is comprised of various secreted and membrane proteins. Our goal was to identify niche molecules participating in stem cell-stem cell and/or stem cell-supporting cell interactions. Here, we isolated genes encoding secreted and membrane proteins from purified male germ stem cells using a signal sequence trap approach. Among the genes identified, we focused on the junctional adhesion molecule 4 (JAM4), an immunoglobulin type cell adhesion molecule. JAM4 protein was actually localized to the plasma membrane in male germ cells. JAM4 expression was downregulated as cells differentiated in both germ cell and hematopoietic cell lineages. To analyze function in vivo, we generated JAM4-deficient mice. Histological analysis of testes from homozygous nulls did not show obvious abnormalities, nor did liver and kidney tissues, both of which strongly express JAM4. The numbers of hematopoietic stem cells in bone marrow were indistinguishable between wild-type and mutant mice, as was male germ cell development. These results suggest that JAM4 is expressed in stem cells and progenitor cells but that other cell adhesion molecules may substitute for JAM4 function in JAM4-deficient mice both in male germ cell and hematopoietic lineages.

Bone morphogenetic protein-2 induces the differentiation of a mesenchymal progenitor cell line, ROB-C26, into mature osteoblasts and adipocytes.

AIMS: Osteoblasts and adipocytes originate from common precursor cells. We examined the effects of bone morphogenetic protein-2 (BMP-2) on the molecular mechanisms governing the diametric actions of BMP-2 on simultaneous mature osteoblast and adipocyte differentiation in a clonal mesenchymal progenitor cell line, ROB-C26 (C26). MAIN METHODS: The present study using RT-PCR, Western blotting and ELISA investigated the effects of BMP-2 on transcription factors for osteoblasts (Runx2, Dlx5, Osterix, Msx2 and AJ18) and adipocytes (PPARgamma2), osteoblastic markers, alkaline phosphatase (ALP), bone sialoprotein (BSP) and osteocalcin (OC), and adipocyte differentiation-dependent protein, aP2 in C26 cells. KEY FINDINGS: BMP-2 increased not only mRNA and protein expressions of Dlx5 followed by inducing Osterix and BSP levels in a dose- and time-dependent manner with an increase in Runx2, Msx2, AJ18, ALP and OC levels, but also induced PPARgamma2 and aP2 levels. At the light microscopic level, BMP-2 induced mature osteoblastic and adipogenic differentiation in the C26 cultures not only by an increase in the number of ALP-positive osteoblasts, in their staining intensity and in the number of mature adipocytes with Oil Red O-positive lipid droplets, but also mineralized matrix formation of the cultures assessed by detecting an increase in calcium concentration and the formation of Alizarin Red S-positive mineralization nodules. SIGNIFICANCE: These results indicate that BMP-2 induces the differentiation of C26 mesenchymal progenitors into mature osteoblasts and adipocytes and the usefulness of this cell line for studying the regulatory mechanism of osteoblast and adipocyte differentiation from common mesenchymal progenitors.

Reducing of salivary α-amylase inhibition by using bovine serum albumin and calcium chloride for forensic saliva screening.

Inhibiting salivary α-amylase is a critical issue of forensic saliva identification using the catalytic method. This study aims to identify human α-amylase inhibitors in forensic saliva screening by using a blue starch amylase test and to measure the extent of enzyme inhibition. Thus, in order to demonstrate the presence of inhibitors, we prepared positively charged metal ion sources or chelators that were mixed into the saliva stains. The results of this study show that ferric chloride (FeCl3), magnesium chloride, ethylenediaminetetraacetic acid, and citric acid significantly decrease the α-amylase activity of saliva stains. We also verified this approach using blood, a magnesium-containing liquid supplement, and two citric acid-containing soft drinks that were contaminated with saliva stains as forensic mock samples; these samples also showed a significant reduction in salivary α-amylase activity. To establish an inhibitor-resistant blue starch amylase test, we applied bovine serum albumin (BSA) and calcium chloride (CaCl2) to the reaction system. The results show that salivary α-amylase inhibition of the forensic mock samples occurred under normal test conditions (i.e., 300ng/µL BSA, 0mM CaCl2), and that inhibition was significantly relieved under the BSA+CaCl2 conditions (i.e., 1000ng/µL BSA, 5mM CaCl2). Therefore, the results of this study demonstrate that both BSA and CaCl2 can be utilized as reaction stabilizers in forensic saliva screening.

The niche for spermatogonial stem cells in the mammalian testis.

The theory of the "stem cell niche" was originally proposed for the hematopoietic system, and the existence of the niche as an actual entity was proved in the Drosophila germ cell system. Historically, mammalian spermatogenesis has been studied extensively as a prime example of a stem cell system, and studies have established a stem-progenitor hierarchical order of spermatogonia. In the niche on the basal lamina of seminiferous tubules, spermatogonial stem cells (SSCs) are secluded from the outside world and divide constantly to self-renew and differentiate. During the last 10 years, the development and exploitation of the germ cell transplantation method has expanded our understanding of the nature of SSCs and their niches. The ability to maintain and expand SSCs in vitro, which recently became possible, has further reinforced this research area as a mecca of stem cell biology. Nonetheless, the mammalian germ stem cell and its niche remain to be defined more strictly and precisely. We are still on a journey in search of the real stem cell and its true niche.

Real-time in vivo imaging of p16Ink4a reveals cross talk with p53.

Expression of the p16(Ink4a) tumor suppressor gene, a sensor of oncogenic stress, is up-regulated by a variety of potentially oncogenic stimuli in cultured primary cells. However, because p16(Ink4a) expression is also induced by tissue culture stress, physiological mechanisms regulating p16(Ink4a) expression remain unclear. To eliminate any potential problems arising from tissue culture-imposed stress, we used bioluminescence imaging for noninvasive and real-time analysis of p16(Ink4a) expression under various physiological conditions in living mice. In this study, we show that oncogenic insults such as ras activation provoke epigenetic derepression of p16(Ink4a) expression through reduction of DNMT1 (DNA methyl transferase 1) levels as a DNA damage response in vivo. This pathway is accelerated in the absence of p53, indicating that p53 normally holds the p16(Ink4a) response in check. These results unveil a backup tumor suppressor role for p16(Ink4a) in the event of p53 inactivation, expanding our understanding of how p16(Ink4a) expression is regulated in vivo.

Stem cell defects in ATM-deficient undifferentiated spermatogonia through DNA damage-induced cell-cycle arrest.

Mammalian spermatogenesis is maintained by stem cell capacity within undifferentiated spermatogonial subpopulation. Here, using a combination of surface markers, we describe a purification method for undifferentiated spermatogonia. Flow cytometric analysis revealed that this population is composed of Plzf-positive cells and exhibits quiescence and the side population phenotype, fulfilling general stem cell criteria. We then applied this method to analyze undifferentiated spermatogonia and stem cell activity of Atm(-/-) mice. Atm(-/-) testis shows progressive depletion of undifferentiated spermatogonia accompanied by cell-cycle arrest. In Atm(-/-) undifferentiated spermatogonia, a self-renewal defect was observed in vitro and in vivo. Accumulation of DNA damage and activation of the p19(Arf)-p53-p21(Cip1/Waf1) pathway were observed in Atm(-/-) undifferentiated spermatogonia. Moreover, suppression of p21(Cip1/Waf1) in an Atm(-/-) background restored transplantation ability of undifferentiated spermatogonia, indicating that ATM plays an essential role in maintenance of undifferentiated spermatogonia and their stem cell capacity by suppressing DNA damage-induced cell-cycle arrest.

Regulation of the self-renewal ability of tissue stem cells by tumor-related genes.

Stem cells are defined by both their ability to produce stem cells, a property known as self-renewal, and their ability to give rise to differentiated progeny. One of the hallmarks of tissue stem cell is its adoption of a non-dividing, undifferentiated state called quiescence. The ability to sustain quiescence is crucial for stem cell function in several tissues. In this review, we discuss how tissue stem cell properties are affected by the products of tumor-related genes such as ATM, PTEN and FOXO. Recent advances in stem cell research achieved using mouse genetics have provided novel evidence that numerous tumor-related genes, which are known to control genomic stability, cell proliferation and survival, are also closely associated with the regulation of tissue stem cell self-renewal. These findings support the notion that tissue stem cells and cancer cells share common properties. Further investigation of stem cell regulation by tumor-related genes may pave the way for successful stem cell-based approaches to regenerative medicine and cancer therapy.

Regulation of reactive oxygen species by Atm is essential for proper response to DNA double-strand breaks in lymphocytes.

The ataxia telangiectasia-mutated (ATM) gene plays a pivotal role in the maintenance of genomic stability. Although it has been recently shown that antioxidative agents inhibited lymphomagenesis in Atm(-/-) mice, the mechanisms remain unclear. In this study, we intensively investigated the roles of reactive oxygen species (ROS) in phenotypes of Atm(-/-) mice. Reduction of ROS by the antioxidant N-acetyl-l-cysteine (NAC) prevented the emergence of senescent phenotypes in Atm(-/-) mouse embryonic fibroblasts, hypersensitivity to total body irradiation, and thymic lymphomagenesis in Atm(-/-) mice. To understand the mechanisms for prevention of lymphomagenesis, we analyzed development of pretumor lymphocytes in Atm(-/-) mice. Impairment of Ig class switch recombination seen in Atm(-/-) mice was mitigated by NAC, indicating that ROS elevation leads to abnormal response to programmed double-strand breaks in vivo. Significantly, in vivo administration of NAC to Atm(-/-) mice restored normal T cell development and inhibited aberrant V(D)J recombination. We conclude that Atm-mediated ROS regulation is essential for proper DNA recombination, preventing immunodeficiency, and lymphomagenesis.

Foxo3a is essential for maintenance of the hematopoietic stem cell pool.

Hematopoietic stem cells (HSCs) are maintained in an undifferentiated quiescent state within a bone marrow niche. Here we show that Foxo3a, a forkhead transcription factor that acts downstream of the PTEN/PI3K/Akt pathway, is critical for HSC self-renewal. We generated gene-targeted Foxo3a(-/-) mice and showed that, although the proliferation and differentiation of Foxo3a(-/-) hematopoietic progenitors were normal, the number of colony-forming cells present in long-term cocultures of Foxo3a(-/-) bone marrow cells and stromal cells was reduced. The ability of Foxo3a(-/-) HSCs to support long-term reconstitution of hematopoiesis in a competitive transplantation assay was also impaired. Foxo3a(-/-) HSCs also showed increased phosphorylation of p38MAPK, an elevation of ROS, defective maintenance of quiescence, and heightened sensitivity to cell-cycle-specific myelotoxic injury. Finally, HSC frequencies were significantly decreased in aged Foxo3a(-/-) mice compared to the littermate controls. Our results demonstrate that Foxo3a plays a pivotal role in maintaining the HSC pool.

Premeiotic germ cell defect in seminiferous tubules of Atm-null testis.

Lifelong spermatogenesis is maintained by coordinated sequential processes including self-renewal of stem cells, proliferation of spermatogonial cells, meiotic division, and spermiogenesis. It has been shown that ataxia telangiectasia-mutated (ATM) is required for meiotic division of the seminiferous tubules. Here, we show that, in addition to its role in meiosis, ATM has a pivotal role in premeiotic germ cell maintenance. ATM is activated in premeiotic spermatogonial cells and the Atm-null testis shows progressive degeneration. In Atm-null testicular cells, differing from bone marrow cells of Atm-null mice, reactive oxygen species-mediated p16(Ink4a) activation does not occur in Atm-null premeiotic germ cells, which suggests the involvement of different signaling pathways from bone marrow defects. Although Atm-null bone marrow undergoes p16(Ink4a)-mediated cellular senescence program, Atm-null premeiotic germ cells exhibited cell cycle arrest and apoptotic elimination of premeiotic germ cells, which is different from p16(Ink4a)-mediated senescence.

Angiopoietin-1 promotes LYVE-1-positive lymphatic vessel formation.

Angiopoietin (Ang) signaling plays a role in angiogenesis and remodeling of blood vessels through the receptor tyrosine kinase Tie2, which is expressed on blood vessel endothelial cells (BECs). Recently it has been shown that Ang-2 is crucial for the formation of lymphatic vasculature and that defects in lymphangiogenesis seen in Ang-2 mutant mice are rescued by Ang-1. These findings suggest important roles for Ang signaling in the lymphatic vessel system; however, Ang function in lymphangiogenesis has not been characterized. In this study, we reveal that lymphatic vascular endothelial hyaluronan receptor 1-positive (LYVE-1(+)) lymphatic endothelial cells (LECs) express Tie2 in both embryonic and adult settings, indicating that Ang signaling occurs in lymphatic vessels. Therefore, we examined whether Ang-1 acts on in vivo lymphatic angiogenesis and in vitro growth of LECs. A chimeric form of Ang-1, cartilage oligomeric matrix protein (COMP)-Ang-1, promotes in vivo lymphatic angiogenesis in mouse cornea. Moreover, we found that COMP-Ang-1 stimulates in vitro colony formation of LECs. These Ang-1-induced in vivo and in vitro effects on LECs were suppressed by soluble Tie2-Fc fusion protein, which acts as an inhibitor by sequestering Ang-1. On the basis of these observations, we propose that Ang signaling regulates lymphatic vessel formation through Tie2.

Spatial analysis of germ stem cell development in Oct-4/EGFP transgenic mice.

Questions persist regarding male germ stem cells and how they mature during the prespermatogenic period of testicular development. We successfully labeled the prespermatogonia with green fluorescence protein (GFP) by using Oct-4 enhancer/promoter. This study shows that GFP was specifically expressed in prespermatogonia, spermatogonia and spermatids that faithfully reproduce the endogenous expression of Oct-4. Histochemical analysis revealed that most of the TRA98-positive gonocytes are also positive for GFP. However, the frequency of GFP expressing cells out of TRA98 expressing cells decreased together with the maturation of gonocytes in the first week after birth. To compare the stem cell activity between GFP-positive and -negative populations, we performed a transplantation of sorted cells into testes from an individual population. Colonization efficiency of germ cells from a GFP-positive population resulted in a 30-fold increase in colonization compared with a GFP-negative population. Since the expression of Oct-4 in prespermtogonia correlates well with the stem activity, Oct-4 might be a crucial molecule in the stem cell property of spermatogonia but not in cell survival.

Derivation and morphological characterization of mouse spermatogonial stem cell lines.

Spermatogonial stem cells (SSCs), having yet to possess decisive markers, can only be detected retrospectively by transplantation assay. It was reported recently that mouse gonocytes collected from DBA/2 and ICR neonates propagated in vitro. This cultured germ cell, named the germline stem cell (GS cell), produced functional sperm to make progeny when transplanted into recipient mouse testes. Here we show that GS cell lines can be established not only from neonatal testes but also from the testis of adult mice. We also confirmed that GS cells once transplanted into a host testis can be recovered to resume in vitro expansion, indicating that they are convertible mutually with SSCs in adult testes. Confocal laser microscopic examination showed GS cells resemble undifferentiated spermatogonia in the adult testis. This unique cell line could be useful for research in germ cell biology and applicable as a new tool for the genetic engineering of animals.

Increment of murine spermatogonial cell number by gonadotropin-releasing hormone analogue is independent of stem cell factor c-kit signal.

Recent studies have demonstrated that GnRH-analogues can stimulate regeneration of spermatogenesis of rats when administered after testicular damages. Although the mechanism of this phenomenon has not been elucidated yet, stem cell factor (SCF) produced by Sertoli cells was proposed to mediate the effects of GnRH-analogues on spermatogonial proliferation and/or survival. In the present study, we quantitatively evaluated the proliferation of spermatogonia and addressed whether SCF mediates the effect of GnRH-analogue on spermatogonial proliferation, using a novel approach combining spermatogonial transplantation and laser confocal microscopic observation. In the first experiment, using wild-type mice as recipients for spermatogonial transplantation, the number of donor spermatogonia per 100 Sertoli cells in each spermatogenic colony was significantly higher in the experimental group of mice treated with leuprorelin, a GnRH-agonist, than that of the control group at 4 and 5 wk after transplantation. In the second experiment, Steel/Steeldickie (Sl/Sld) mutant mice, which lack expression of membrane bound form SCF, were used as recipients. As seen in the first experiment, the number of undifferentiated spermatogonia was significantly higher in leuprorelin-treated than in the control group. Since undifferentiated spermatogonia do not express the receptor of SCF, the present study clearly demonstrates that neither membrane-bound nor secreted forms of SCF are involved in the mechanism of GnRH-analogue's effect on spermatogonial proliferation and/or survival.

Expansion of murine spermatogonial stem cells through serial transplantation.

Mammalian male germ cells might be generally thought to have infinite proliferative potential based on their life-long production of huge numbers of sperm. However, there has been little substantial evidence that supports this assumption. In the present study, we performed serial transplantation of spermatogonial stem cells to investigate if they expand by self-renewing division following transplantation. The transgenic mouse carrying the Green fluorescent protein gene was used as the donor cell source that facilitated identification and recollection of colonized donor germ cells in the recipient testes. The established colonies of germ cells in the recipient testes were collected and transplanted to new recipients. This serial transplantation of spermatogonial stem cells repopulated the recipient testes, which were successfully performed sequentially up to four times from one recipient to the next. The incubation periods between two sequential transplantations ranged from 55 to 373 days. During these passages, the spermatogonial stem cells showed constant activity to form spermatogenic colonies in the recipient testis. They continued to increase in number for more than a year following transplantation. Colonization efficiency of spermatogonial stem cells was determined to be 4.25% by using Sl/Sl(d) mice as recipients that propagated only undifferentiated type A spermatogonia in their testes. Based on the colonization efficiency, one colony-forming activity was assessed to equate to about 20 spermatogonial stem cells. The spermatogonial stem cells were estimated to expand over 50-fold in 100 days in this experiment.

Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche.

The quiescent state is thought to be an indispensable property for the maintenance of hematopoietic stem cells (HSCs). Interaction of HSCs with their particular microenvironments, known as the stem cell niches, is critical for adult hematopoiesis in the bone marrow (BM). Here, we demonstrate that HSCs expressing the receptor tyrosine kinase Tie2 are quiescent and antiapoptotic, and comprise a side-population (SP) of HSCs, which adhere to osteoblasts (OBs) in the BM niche. The interaction of Tie2 with its ligand Angiopoietin-1 (Ang-1) induced cobblestone formation of HSCs in vitro and maintained in vivo long-term repopulating activity of HSCs. Furthermore, Ang-1 enhanced the ability of HSCs to become quiescent and induced adhesion to bone, resulting in protection of the HSC compartment from myelosuppressive stress. These data suggest that the Tie2/Ang-1 signaling pathway plays a critical role in the maintenance of HSCs in a quiescent state in the BM niche.

Identification and characterization of stem cells in prepubertal spermatogenesis in mice.

The stem cell properties of gonocytes and prospermatogonia at prepubertal stages are still largely unknown: it is not clear whether gonocytes and prospermatogonia are a special cell type or similar to adult undifferentiated spermatogonia. To characterize these cells, we have established transgenic mice carrying EGFP (enhanced green fluorescence protein) cDNA under control of an Oct4 18-kb genomic fragment containing the minimal promoter and proximal and distal enhancers; Oct4 is reported to be expressed in undifferentiated spermatogonia at prepubertal stages. Generation of transgenic mice enabled us to purify gonocytes and prospermatogonia from the somatic cells of the testis. Transplantation studies of testicular cells so far have been done with a mixture of germ cells and somatic cells. This is the first report that establishes how to purify germ cells from total testicular cells, enabling evaluation of cell-autonomous repopulating activity of a subpopulation of prospermatogonia. We show that prospermatogonia differ markedly from adult spermatogonia in both the size of the KIT-negative population and cell cycle characteristics. The GFP(+) KIT(-) fraction of prospermatogonia has much higher repopulating activity than does the GFP(+)KIT(+) population in the adult environment. Interestingly, the GFP(+)KIT(+) population still exhibits repopulating activity, unlike adult KIT-positive spermatogonia. We also show that ALCAM, activated leukocyte cell adhesion molecule, is expressed transiently in gonocytes. Sertoli cells and myoid cells also express ALCAM at the same stage, suggesting that ALCAM may contribute to gonocyte-Sertoli cell adhesion and migration of gonoyctes toward the basement membrane.