Effective enrichment of stem cells in regenerating Xenopus laevis tadpole tails using the side population method.

Xenopus laevis tadpoles have a strong regenerative ability and can regenerate their whole tails after tail amputation. Lineage-restricted tissue stem cells are thought to provide sources for the regenerating tissues by producing undifferentiated progenitor cells in response to tail amputation. However, elucidating the behavioral dynamics of tissue stem cells during tail regeneration is difficult because of their rarity, and there are few established methods of isolating these cells in amphibians. Here, to detect and analyze rare tissue stem cells, we attempted to enrich tissue stem cells from tail regeneration buds. High Hoechst dye efflux capacity is thought to be a common characteristic of several types of mammalian tissue stem cells; these stem cells, designated as the "side population (SP)," may be enriched by flow cytometry (SP method). To evaluate the effectiveness of stem cell enrichment using the SP method in regenerating X. laevis tadpole tails, we performed single-cell RNA sequencing (scRNA-seq) of SP cells from regeneration buds and analyzed the frequency of satellite cells, which are muscle stem/progenitor cells expressing pax7. The pax7-expressing cells were enriched in the SP compared with whole normal tails and regeneration buds. Furthermore, hes1-expressing cells, which are assumed to be neural stem/progenitor cells, were also enriched in the SP. Our findings suggest that the SP method is efficient for successfully enriching tissue stem cells in regenerating X. laevis tadpole tails, indicating that the combination of the SP method and scRNA-seq is useful for studying tissue stem cells that contribute to tail regeneration.

Combination Treatment with Human Adipose Tissue Stem Cell-derived Exosomes and Fractional CO2 Laser for Acne Scars: A 12-week Prospective, Double-blind, Randomized, Split-face Study.

A variety of applications of human adipose tissue stem cell-derived exosomes have been suggested as novel cell-free therapeutic strategies in the regenerative and aesthetic medical fields. This study evaluated the clinical efficacy and safety of adipose tissue stem cell-derived exosomes as an adjuvant therapy after application of fractional CO2 laser for acne scars. A 12-week prospective, double-blind, randomized, split-face trial was performed. A total of 25 patients received 3 consecutive treatment sessions of fractional CO2 laser to the whole face, with a follow-up evaluation. Post-laser treatment regimens were applied; for each patient, one side of the face was treated with adipose tissue stem cell-derived exosomes gel and the other side was treated with control gel. Adipose tissue stem cell-derived exosomes-treated sides had achieved a significantly greater improvement than the control sides at the final follow-up visit (percentage reduction in échelle d'évaluation clinique des cicatrices d'acné scores: 32.5 vs 19.9%, p < 0.01). Treatment-related erythema was milder, and post-treatment downtime was shorter on the applications of human adipose tissue stem cell-derived exosomes-treated side. In conclusion, the combined use of this novel material with resurfacing devices would provide synergistic effects on both the efficacy and safety of atrophic acne scar treatments.

Transplantation of Airway Epithelial Stem/Progenitor Cells: A Future for Cell-Based Therapy.

Cell therapy has the potential to cure disease through replacement of malfunctioning cells. Although the tissue stem cell (TSC) is thought to be the optimal therapeutic cell, transplantation of TSC/progenitor cell mixtures has saved lives. We previously purified the mouse tracheobronchial epithelial TSCs and reported that in vitro amplification generated numerous TSCs. However, these cultures also contained TSC-derived progenitor cells and TSC repurification by flow cytometry compromised TSC self-renewal. These limitations prompted us to determine if a TSC/progenitor cell mixture would repopulate the injured airway epithelium. We developed a cell transplantation protocol and demonstrate that transplanted mouse and human tracheobronchial epithelial TSC/progenitor cell mixtures are 20-25% of airway epithelial cells, actively contribute to epithelial repair, and persist for at least 43 days. At 2 weeks after transplantation, TSCs/progenitor cells differentiated into the three major epithelial cell types: basal, secretory, and ciliated. We conclude that cell therapy that uses adult tracheobronchial TSCs/progenitor cells is an effective therapeutic option.

Functional roles of Lgr4 and Lgr5 in embryonic gut, kidney and skin development in mice.

Lgr4 and Lgr5 are known markers of adult and embryonic tissue stem cells in various organs. However, whether Lgr4 and Lgr5 are important for embryonic development remains unclear. To study their functions during intestinal crypt, skin and kidney development we now generated mice lacking either Lgr4 (Lgr4KO), Lgr5 (Lgr5KO) or both receptors (Lgr4/5dKO). E16.5 Lgr4KO mice displayed complete loss of Lgr5+/Olfm4+intestinal stem cells, compromised Wnt signaling and impaired proliferation and differentiation of gut epithelium. Similarly, E16.5 Lgr4KO mice showed reduced basal cell proliferation and hair follicle numbers in the developing skin, as well as dilated kidney tubules and ectatic Bowman׳s spaces. Although Lgr4KO and Lgr5KO mice both died perinatally, Lgr5 deletion did not compromise embryonic development of gut, kidney or skin. Concomitant deletion of Lgr4 and Lgr5 did not prevent perinatal lethality, in contrast to a previous report that suggested rescue of Lgr5 KO perinatal lethality by a hypomorphic Lgr4 mutant. While the double deletion did not further promote the phenotypes observed in Lgr4KO intestines, impaired kidney cell proliferation, reduced epidermal thickness, loss of Lgr5+follicular epithelium and impaired hair follicle development were only observed in Lgr4/5dKO mice. This supports complementary functions of both receptors. Our findings clearly establish the importance of Lgr4 and Lgr5 during embryonic gut, skin and kidney development, with a dominant role of Lgr4.

Origin of Vocal Fold Stellate Cells in the Human Macula Flava.

OBJECTIVES: There is growing evidence that vocal fold stellate cells (VFSCs) in the human maculae flavae are tissue stem cells of the human vocal fold and that the maculae flavae are a stem cell niche. The origin of the cells in the human maculae flavae (CHMF) and the relationship with bone marrow-derived cells were investigated. METHODS: Five human adult vocal fold mucosae were investigated. The CHMF were subcultured and morphological features were assessed. Immunoreactivity to antibodies directed to cytokeratin, desmin, GFAP, vimentin, CD34, CD45, and collagen type I was investigated. RESULTS: Cultured CHMF formed a colony-forming unit, indicating they are mesenchymal stem cells or stromal stem cells in the bone marrow. The CHMF expressed hematopoietic markers (CD34, CD45) and collagen type I, which are the major makers for bone marrow-derived circulating fibrocytes. The cultured CHMF expressed epithelium-associated, muscle-associated, neural-associated, and mesenchymal cell-associated proteins, indicating the CHMF are undifferentiated and express proteins of all 3 germ layers. CONCLUSIONS: The CHMF are undifferentiated cells derived from the differentiation of bone marrow cells. The results of this study are consistent with the hypothesis that the VFSCs are tissue stem cells or progenitor cells of the human vocal fold mucosa.

A Kinetic Stem Cell Counting Analysis of the Specific Effects of Cell Culture Medium Growth Factors on Adipose-Derived Mesenchymal Stem Cells.

A recently described kinetic stem cell (KSC) counting method was used to investigate the stem-cell-specific effects of commercial growth factor supplements used for expanding stem cells in adipose-tissue-derived mesenchymal cell preparations. The supplements were a proprietary growth factor product, a source of fetal bovine serum, two sources of pooled human sera, and two sources of human platelet lysate. KSC counting analyses were performed to monitor effects on the fraction and viability of stem cells in serial cultures with their respective supplements. Serial cultures supplemented with the proprietary growth factor product or fetal bovine serum showed a similar high degree of maintenance of stem cell fraction with passage. In contrast, cultures supplemented with human sera or human platelet lysate showed rapid declines in stem cell fraction. KSC counting was used to discover the cellular basis for the decreasing stem cell fractions. For human platelet lysate, it was attributable to lower rates of self-renewing symmetric stem cell divisions. For human sera, both low rates of symmetric division and high rates of stem cell death were responsible. These results demonstrate the power of the KSC counting method to provide previously inaccessible information for improving future tissue stem cell biomanufacturing.

Evaluation of the Precision of Kinetic Stem Cell (KSC) Counting for Specific Quantification of Human Mesenchymal Stem Cells in Heterogeneous Tissue Cell Preparations.

Kinetic stem cell (KSC) counting is a recently introduced first technology for quantifying tissue stem cells in vertebrate organ and tissue cell preparations. Previously, effective quantification of the fraction or dosage of tissue stem cells had been largely lacking in stem cell science and medicine. A general method for the quantification of tissue stem cells will accelerate progress in both of these disciplines as well as related industries like drug development. Triplicate samples of human oral alveolar bone cell preparations, which contain mesenchymal stem cells (MSCs), were used to estimate the precision of KSC counting analyses conducted at three independent sites. A high degree of intra-site precision was found, with coefficients of variation for determinations of MSC-specific fractions of 8.9% (p < 0.003), 13% (p < 0.006), and 25% (p < 0.02). The estimates of inter-site precision, 11% (p < 0.0001) and 26% (p < 0.0001), also indicated a high level of precision. Results are also presented to show the ability of KSC counting to define cell subtype-specific kinetics factors responsible for changes in the stem cell fraction during cell culture. The presented findings support the continued development of KSC counting as a new tool for advancing stem cell science and medicine.

Thymic stromal lymphopoietin controls hair growth.

Skin tissue regeneration after injury involves the production and integration of signals by stem cells residing in hair follicles (HFSCs). Much remains unknown about how specific wound-derived factors modulate stem cell contribution to hair growth. We demonstrate that thymic stromal lymphopoietin (TSLP) is produced in response to skin injury and during the anagen phase of the hair cycle. Intradermal injection of TSLP promoted wound-induced hair growth (WIHG), whereas neutralizing TSLP receptor (TSLPR) inhibited WIHG. Using flow cytometry and fluorescent immunostaining, we found that TSLP promoted proliferation of transit-amplifying cells. Lgr5CreER-mediated deletion of Tslpr in HFSCs inhibited both wound-induced and exogenous TSLP-induced hair growth. Our data highlight a novel function for TSLP in regulation of hair follicle activity during homeostasis and wound healing.

Periodontal tissue stem cells and mesenchymal stem cells in the periodontal ligament.

Periodontal tissue stem cells, which play a crucial role in maintaining the homeostasis of periodontal tissues, are found in the periodontal ligament (PDL). These cells have long been referred to as mesenchymal stem/stromal cells (MSCs), and their clinical applications have been extensively studied. However, tissue stem cells in the PDL have not been thoroughly investigated, and they may be different from MSCs. Recent advances in stem cell biology, such as genetic lineage tracing, identification of label-retaining cells, and single-cell transcriptome analysis, have made it possible to analyze tissue stem cells in the PDL in vivo. In this review, we summarize recent findings on these stem cell populations in PDL and discuss future research directions toward developing periodontal regenerative therapy.

Stroke and Neurogenesis: Bridging Clinical Observations to New Mechanistic Insights from Animal Models.

Stroke was the 2nd leading cause of death and a major cause of morbidity. Unfortunately, there are limited means to promote neurological recovery post-stroke, but research has unearthed potential targets for therapies to encourage post-stroke neurogenesis and neuroplasticity. The occurrence of neurogenesis in adult mammalian brains, including humans, was not widely accepted until the 1990s. Now, adult neurogenesis has been extensively studied in human and mouse neurogenic brain niches, of which the subventricular zone of the lateral ventricles and subgranular zone of the dentate gyrus are best studied. Numerous other niches are under investigation for neurogenic potential. This review offers a basic overview to stroke in the clinical setting, a focused summary of recent and foundational research literature on cortical neurogenesis and post-stroke brain plasticity, and insights regarding how the meninges and choroid plexus have emerged as key players in neurogenesis and neuroplasticity in the context of focal cerebral ischemia disrupting the anterior circulation. The choroid plexus and meninges are vital as they are integral sites for neuroimmune interactions, glymphatic perfusion, and niche signaling pertinent to neural stem cells and neurogenesis. Modulating neuroimmune interactions with a focus on astrocyte activity, potentially through manipulation of the choroid plexus and meningeal niches, may reduce the exacerbation of stroke by inflammatory mediators and create an environment conducive to neurorecovery. Furthermore, addressing impaired glymphatic perfusion after ischemic stroke likely supports a neurogenic environment by clearing out inflammatory mediators, neurotoxic metabolites, and other accumulated waste. The meninges and choroid plexus also contribute more directly to promoting neurogenesis: the meninges are thought to harbor neural stem cells and are a niche amenable to neural stem/progenitor cell migration. Additionally, the choroid plexus has secretory functions that directly influences stem cells through signaling mechanisms and growth factor actions. More research to better understand the functions of the meninges and choroid plexus may lead to novel approaches for stimulating neuronal recovery after ischemic stroke.

Single or Repeated Ablation of Mouse Olfactory Epithelium by Methimazole.

Odor-detecting olfactory sensory neurons residing in the nasal olfactory epithelium (OE) are the only neurons in direct contact with the external environment. As a result, these neurons are subjected to chemical, physical, and infectious insults, which may be the underlying reason why neurogenesis occurs in the OE of adult mammals. This feature makes the OE a useful model for studying neurogenesis and neuronal differentiation, with the possibility for systemic as well as local administration of various compounds and infectious agents that may interfere with these cellular processes. Several different chemical compounds have been shown to cause toxic injury to the OE, which can be used for OE ablation. We, and others, have found that the systemic administration of the hyperthyroid drug, methimazole, reliably causes olfactotoxicity as a side effect. Here, we outline an OE lesioning protocol for single or repeated ablation by methimazole. A single methimazole administration can be used to study neuroepithelial regeneration and stem cell activation, while repeated ablation and regeneration of OE enable the study of tissue stem cell exhaustion and generation of tissue metaplasia.

Adipose-Derived Stem Cell: "Treat or Trick".

Stem cells have been widely used for treating disease due to the various benefits they offer in the curing process. Several treatments using stem cells have undergone clinical trials, such as cell-based therapies for heart disease, sickle cell disease, thalassemia, etc. Adipose-derived stem cells are some of the many mesenchymal stem cells that exist in our body that can be harvested from the abdomen, thighs, etc. Adipose tissue is easy to harvest, and its stem cells can be obtained in higher volumes compared to stem cells harvested from bone marrow, for which a more invasive technique is required with a smaller volume obtained. Many scientists have expressed interest in investigating the role of adipose-derived stem cells in treating disease since their use was first described. This is due to these stem cells' ability to differentiate into multiple lineages and secrete a variety of growth factors and proteins. Previous studies have found that the hormones, cytokines, and growth factors contained in adipose tissue play major roles in the metabolic regulation of adipose tissue, as well as in energy balance and whole-body homeostasis through their endocrine, autocrine, and paracrine functions. These are thought to be important contributors to the process of tissue repair and regeneration. However, it remains unclear how effective and safe ADSCs are in treating diseases. The research that has been carried out to date is in order to investigate the impact of ADSCs in disease treatment, as described in this review, to highlight its "trick or treat" effect in medical treatment.

Characterization of Crystalline Phase of TiO2 Nanocrystals, Cytotoxicity and Cell Internalization Analysis on Human Adipose Tissue-Derived Mesenchymal Stem Cells.

Titanium dioxide (TiO2) is manufactured worldwide as crystalline and amorphous forms for multiple applications, including tissue engineering, but our study proposes analyzing the impact of crystalline phases of TiO2 on Mesenchymal Stem Cells (MSCs). Several studies have already described the regenerative potential of MSCs and TiO2 has been used for bone regeneration. In this study, polydispersity index and sizes of TiO2 nanocrystals (NCs) were determined. Adipose tissue-derived Mesenchymal Stem Cells (AT-MSCs) were isolated and characterized in order to evaluate cellular viability and the internalization of nanocrystals (NCs). All of the assays were performed using the TiO2 NCs with 100% anatase (A), 91.6% anatase/9.4% rutile (AR), 64.6% rutile/35.4% anatase (RA), and 84.0% rutile/16% brookite (RB), submitted to several concentrations in 24-h treatments. Cellular localization of TiO2 NCs in the AT-MSCs was resolved by europium-doped NCs. Viability was significantly improved under the predominance of the rutile phase in NCs with localization restricted at the cytoplasm, suggesting that AR and RA NCs are not genotoxic and can be associated with most cellular activities and metabolic pathways, including glycolysis and cell division.

Heterogeneity and hierarchy of the tissue stem cells in the human newborn vocal fold mucosa.

OBJECTIVES: There is growing evidence that the cells in the maculae flavae (MFe) are tissue stem cells and the MFe are a stem cell niche of the human vocal fold mucosa. Heterogeneity and hierarchy of tissue stem cells in the MFe of newborn vocal fold were investigated in vivo. STUDY DESIGN: Histologic analysis of the human vocal folds. METHODS: Five normal human newborn vocal folds were investigated under transmission electron microscopy and light microscopy. RESULTS: Cobblestone-like polygonal cells, vocal fold stellate cell-like cells, and fibroblast-like spindle cells were intermingled in the newborn MFe in vivo, indicating that the cells in the MFe had heterogeneity. However, cobblestone-like polygonal cells were predominant. Free ribosomes were well developed in the cytoplasm. The cells in some cases formed gap junctions with each other. The cells in some cases were attached to other cells and formed cell junctions with each other. These findings indicated cells in the newborn maculae flavae possessed features of mesenchymal cells (cells in mesenchyme). Colony-forming-unit-like cell aggregate was observed, indicating the cells in the newborn MFe had stemness. The cobblestone-like polygonal cells expressed SSEA-3 (a human pluripotent stem cell marker), indicating they were at the top of a cellular hierarchy in the stem cell system. CONCLUSIONS: The cells in the MFe of the human newborn vocal fold mucosa had heterogeneity and hierarchy in the stem cell system in vivo. At birth, newborn maculae flavae are ready to start the growth of the vocal fold mucosa as a vibrating tissue.

SERK Receptor-like Kinases Control Division Patterns of Vascular Precursors and Ground Tissue Stem Cells during Embryo Development in Arabidopsis.

During embryo development, the vascular precursors and ground tissue stem cells divide to renew themselves and produce the vascular tissue, endodermal cells, and cortical cells. However, the molecular mechanisms regulating division of these stem cells have remained largely elusive. In this study, we show that loss of function of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) genes results in aberrant embryo development. Fewer cortical, endodermal, and vascular cells are generated in the embryos of serk1 serk2 bak1 triple mutants. WUSCHEL-RELATED HOMEOBOX 5 (WOX5) is ectopically expressed in vascular cells of serk1 serk2 bak1 embryos. The first transverse division of vascular precursors in mid-globular embryos and second asymmetric division of ground tissue stem cells in early-heart embryos are abnormally altered to a longitudinal division. The embryo defects can be partially rescued by constitutively activated mitogen-activated protein kinase (MAPK) kinase kinase YODA (YDA) and MAPK kinase MKK5. Taken together, our results reveal that SERK-mediated signals regulate division patterns of vascular precursors and ground tissue stem cells, likely via the YDA-MKK4/5 cascade, during embryo development.

Isolation of Bipotential Liver Progenitor Cells from Neonatal Mouse Liver.

Liver stem/progenitor cells (LPCs) are defined as bipotential cells differentiating into both hepatocytes and cholangiocytes. The Notch, TGFß, and Hippo pathways have been implicated in lineage determination of LPCs during development and regeneration. However, the molecular mechanisms governing the lineage specification have not been fully elucidated, yet. Epithelial adhesion molecule (EpCAM) is a marker of cholangiocytes and of LPCs. We found that EpCAM+ cells isolated from neonatal liver contain LPCs that clonally proliferate and are bipotential in vitro and in vivo. Furthermore, EpCAM+ progenies keep the capacity of bidirectional differentiation even after long-term culture. These cells are useful to investigate the molecular mechanisms regulating lineage commitment and epithelial differentiation of LPCs.

A basic study on self-reconstitution of alveolar epithelium-like cells by tissue stem cells in mouse lung.

In recent research on regenerative medicine, three-dimensional (3D) tissue reconstruction using the induced pluripotent stem cell (iPS cell) differentiated cells has attracted attention. In this study, mouse lungs at 1.5, 10, and 20 d old were subjected to enzyme treatment, and aggregates formed in serum-free suspension culture (3D-culture) were observed. The number of aggregates formed was the highest in 1.5 d. The cell aggregates in which the interior of the aggregate is filled and form small vacuoles and the organoid-like aggregates having a relatively large vacuole inside and forming the alveolar-like structure were observed. At 1.5 d, the formation ratio of the organoid-like aggregates was the highest and aggregate size was small at 20 d. For the cell aggregates derived from 1.5 d, positive cells of SSEA-1, CD29, CD90, CD105, alveolar epithelial stem cell marker of SP-C, and Sca-1 were observed in the center. In the cell aggregates derived from 10 d, the expression level of 1.5 d each protein markers and OCT4 gene of transcription factor was decreased, and furthermore, markers were hardly observed in the organoid-like aggregates derived from 10 d. In addition, cells surrounding the vacuole of organoid-like aggregate obtained over 10 d differentiated into periodic acid-Schiff (PAS), podoplanin-positive cells. When the formed cell aggregates were dispersed, cell aggregates and organoid-like aggregates were reformed. Comparing 3D-culture and adhesion culture (2D-culture), SP-C expression of 10 d of cells was maintained. Expression of markers of undifferentiated markers and alveolar tissue stem cells decreased when cell aggregates were cultured with the addition of fetal bovine serum.

Human Adipose Tissue Stem Cells Promote the Growth of Acute Lymphoblastic Leukemia Cells in NOD/SCID Mice.

In this study, the effect of adipose tissue stem cells (ASCs) on the growth of acute lymphoblastic leukemia (ALL) cells was examined in an in vivo model. We established ALL cell lines expressing firefly luciferase (ALL/fLuc) by lentiviral infection that were injected intraperitoneally to NOD/SCID mice. The luciferase activities were significantly higher in mice co-injected with 105 ALL/fLuc cells and ASCs than in those injected with ALL/fLuc cells alone. Co-injection of 105 ALL/fLuc cells and ASCs in differing ratios into mice gradually increased the bioluminescence intensity in all groups, and mice co-injected with 1 or 2 × 106 ASCs showed higher bioluminescence intensity than those receiving lower numbers. Interestingly, in the mice injected with 105 or 107 ALL/fLuc cells alone, the formation of tumor masses was not observed for at least five weeks. Moreover, co-injection of 107 ALL/fLuc cells and 5 × 105 ASCs into mice increased the bioluminescence intensity in all groups, and showed significantly higher bioluminescence intensity compared to mice co-injected with human normal fibroblast HS68 cells. Overall, ASCs promote the growth of ALL cells in vivo, suggesting that ASCs negatively influence hematologic malignancy, which should be considered in developing cell therapy using ASCs.

Long-term organoid culture reveals enrichment of organoid-forming epithelial cells in the fimbrial portion of mouse fallopian tube.

A recent paradigm shift in ovarian cancer research is the finding that many ovarian cancers may originate from fallopian tube epithelial (FTE) cells. As tissue stem and progenitor cells often serve as cells of origin of cancer, a better understanding of FTE stem/progenitor cells and how they become transformed is essential for early detection and prevention of ovarian cancer. To facilitate study of FTE stem/progenitor cells in model systems, we established an organoid culture system for mouse FTE cells. We find that EPCAM+ mouse FTE cells can be stably cultured long-term under a minimal condition of activated EGF signaling and suppressed TGFbeta signaling. We show that both Notch and Wnt signaling are required for growth of FTE cells in organoids, and further activation of Wnt signaling supports their maturation toward the ciliated cell lineage. Lastly, by analyzing the frequency of organoid-forming cells in different portions of the fallopian tube (FT), we find that the distal portion of the FT, which includes the fimbria, is enriched with organoid-forming FTE stem cells.

Effects of dose rates on radiation-induced replenishment of intestinal stem cells determined by Lgr5 lineage tracing.

An understanding of the dynamics of intestinal Lgr5(+) stem cells is important for elucidating the mechanism of colonic cancer development. We previously established a method for evaluating Lgr5(+) stem cells by tamoxifen-dependent Lgr5-lineage tracing and showed that high-dose-rate radiation stimulated replenishment of colonic stem cells. In this study, we evaluated the effects of low-dose-rate radiation on stem cell maintenance. Tamoxifen (4OHT)-injected Lgr5-EGFP-IRES-Cre(ERT2) × ROSA-LSL-LacZ mice were used, LacZ-labeled colonic crypts were enumerated, and the loss of LacZ(+) crypts under low-dose-rate radiation was estimated. After 4OHT treatment, the number of LacZ-labeled Lgr5(+) stem cells was higher in the colon of infant mice than in adult mice. The percentage of LacZ-labeled crypts in infant mice rapidly decreased after 4OHT treatment. However, the percentage of labeled crypts plateaued at ∼2% at 4 weeks post-treatment and remained unchanged for up to 7 months. Thus, it will be advantageous to evaluate the long-term effects of low-dose-rate radiation. Next, we determined the percentages of LacZ-labeled crypts irradiated with 1 Gy administered at different dose rates. As reported in our previous study, mice exposed to high-dose-rate radiation (30 Gy/h) showed a marked replenishment (P = 0.04). However, mice exposed to low-dose-rate radiation (0.003 Gy/h) did not exhibit accelerated stem-cell replenishment (P = 0.47). These findings suggest the percentage of labeled crypts can serve as a useful indicator of the effects of dose rate on the stem cell pool.