scholarly journals Neonatal Wnt-dependent Lgr5 positive stem cells are essential for uterine gland development

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Ryo Seishima ◽  
Carly Leung ◽  
Swathi Yada ◽  
Katzrin Bte Ahmed Murad ◽  
Liang Thing Tan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Reproductive Tract ◽  
Ex Vivo ◽  
Lineage Tracing ◽  
Major Advance ◽  
Uterine Gland ◽  
And Function ◽  
Gland Development

AbstractWnt signaling is critical for directing epithelial gland development within the uterine lining to ensure successful gestation in adults. Wnt-dependent, Lgr5-expressing stem/progenitor cells are essential for the development of glandular epithelia in the intestine and stomach, but their existence in the developing reproductive tract has not been investigated. Here, we employ Lgr5-2A-EGFP/CreERT2/DTR mouse models to identify Lgr5-expressing cells in the developing uterus and to evaluate their stem cell identity and function. Lgr5 is broadly expressed in the uterine epithelium during embryogenesis, but becomes largely restricted to the tips of developing glands after birth. In-vivo lineage tracing/ablation/organoid culture assays identify these gland-resident Lgr5high cells as Wnt-dependent stem cells responsible for uterine gland development. Adjacent Lgr5neg epithelial cells within the neonatal glands function as essential niche components to support the function of Lgr5high stem cells ex-vivo. These findings constitute a major advance in our understanding of uterine development and lay the foundations for investigating potential contributions of Lgr5+ stem/progenitor cells to uterine disorders.

scholarly journals Efficient Ex Vivo Expansion of Highly Purified Human Umbilical Cord Blood-Derived Very Small CD34+lin-CD45- Stem Cells into Functional Endothelial Cells in Vitro in Chemically Identified, Feeder Layer-Free Medium Supplemented with Nicotinamide

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4882-4882
Author(s):  
Alison Domingues ◽  
Kamila Bujko ◽  
Magdalena Kucia ◽  
Janina Ratajczak ◽  
Mariusz Z Ratajczak
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Cell Therapy ◽  
Progenitor Cells ◽  
Umbilical Cord Blood ◽  
Ex Vivo ◽  
Germ Layer ◽  
Differentiation Factors

Background . There is an ongoing search for multipotent stem cells from umbilical cord blood (UCB) with trans-germ layer differentiation potential that can be employed in repairing damaged organs and also expanded into transplantable hematopoietic stem cells (HSCs) and endothelial progenitor cells (EPCs). The existence of such cells in postnatal life could also revive the concept of hemangioblasts or hemangioblast-like cells in adult hematopoietic organs. Our group was the first to isolate a population of small CD34+CD133+lin-CD45- early-development stem cells from human hematopoietic tissues, including UCB. Based on the validated expression of early-development markers, these cells were named "very small embryonic-like stem cells" (VSELs, Circulation Res 2019; 124:208-210). Currently, more than 25 independent groups worldwide who have carefully followed the multicolor-staining cell-sorting strategy described by us (Current Protocols in Cytometry 2010, 9.29.1-9.29.15) have successfully isolated these cells and demonstrated their in vivo contribution to all three germ layer lineages. Thus, VSELs could be very useful in regenerative medicine in the field of angiogenesis, and UCB is an attractive source, with easy accessibility and tolerance to allogenic grafts. However, the low number of these cells in UCB and their quiescence are limiting factors. Therefore, in vitro differentiation of VSELs into endothelial progenitor cells (EPCs) would allow improvement in the ability to expand endothelial cells and could represent a clinically relevant alternative to embryonic stem cells (ESCs) and induced pluripotent stem cells (iPS) for cell therapy without ethical problems and undesirable side effects. Hypothesis. We hypothesized that UCB-purified, very small, early-developmentCD34+lin-CD45-stem cells can be ex vivo expanded into functional EPCs. Materials and Methods. VSELs highly purified by FACS were expanded into EPCs in pro-angiogenic medium supplemented with mesodermic differentiation factors and then endothelial differentiation factors in the presence of nicotinamide and UM171. In parallel, we expanded EPCs from MNCs isolated from the same UCB units by employing a classical protocol (Methods in Enzymology 2008, 445:303-29). The EPC nature of the expanded VSEL-derived cells was confirmed by the expression of typical EPC markers as well as by in vitro angiogenic assays. Results. Our differentiation cocktail allowed us to differentiate and expand VSELs into EPCs. In our expansion medium (Figure 1), the very small, round VSELs smaller than 6 mm in diameter proliferated and differentaited over time into larger and extended cells with a cobblestone morphology similar to the EPC control cells, and we confirmed their endothelial characteristics by cytometry analysis. Like EPCs, VSEL-derived EPCs were positive for CD31, CD144, KDR, and CD105 and negative for mesenchymal surface markers, such as CD90. They also performed similarly to EPCs in classical vasculogenic tests, including adhesion, proliferation, migration, and tubulogenesis assays. Conclusions. This work shows, for the first time, efficient VSEL differentiation into functional endothelial cells with vasculogenic properties without the help of co-culture over feeder-layers or viral vectors in medium supplemented with nicotinamide and UM171. These findings allow us to propose these cells as an interesting cell therapy product. These results also reopen the question of the existence of hemangioblast-like cells in postnatal tissues. We are currently testing these cells in vivo in model of hind limb ischemia. Figure 1 Disclosures No relevant conflicts of interest to declare.

scholarly journals Stem cells and lung regeneration

2020 ◽  
Vol 319 (4) ◽  
pp. C675-C693
Author(s):  
Kalpaj R. Parekh ◽  
Janna Nawroth ◽  
Albert Pai ◽  
Shana M. Busch ◽  
Christiana N. Senger ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Lung Disease ◽  
Progenitor Cell ◽  
Human Lung ◽  
Ex Vivo ◽  
Cell Types ◽  
And Function ◽  
Lung Regeneration

The ability to replace defective cells in an airway with cells that can engraft, integrate, and restore a functional epithelium could potentially cure a number of lung diseases. Progress toward the development of strategies to regenerate the adult lung by either in vivo or ex vivo targeting of endogenous stem cells or pluripotent stem cell derivatives is limited by our fundamental lack of understanding of the mechanisms controlling human lung development, the precise identity and function of human lung stem and progenitor cell types, and the genetic and epigenetic control of human lung fate. In this review, we intend to discuss the known stem/progenitor cell populations, their relative differences between rodents and humans, their roles in chronic lung disease, and their therapeutic prospects. Additionally, we highlight the recent breakthroughs that have increased our understanding of these cell types. These advancements include novel lineage-traced animal models and single-cell RNA sequencing of human airway cells, which have provided critical information on the stem cell subtypes, transition states, identifying cell markers, and intricate pathways that commit a stem cell to differentiate or to maintain plasticity. As our capacity to model the human lung evolves, so will our understanding of lung regeneration and our ability to target endogenous stem cells as a therapeutic approach for lung disease.

scholarly journals The Elusive Endometrial Epithelial Stem/Progenitor Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Fiona L. Cousins ◽  
Ronald Pandoy ◽  
Shiying Jin ◽  
Caroline E. Gargett
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Potential Role ◽  
Lineage Tracing ◽  
Regenerative Capacity ◽  
Proliferative Phase ◽  
Functional Assays ◽  
Human And Mouse

The human endometrium undergoes approximately 450 cycles of proliferation, differentiation, shedding and regeneration over a woman’s reproductive lifetime. The regenerative capacity of the endometrium is attributed to stem/progenitor cells residing in the basalis layer of the tissue. Mesenchymal stem cells have been extensively studied in the endometrium, whereas endometrial epithelial stem/progenitor cells have remained more elusive. This review details the discovery of human and mouse endometrial epithelial stem/progenitor cells. It highlights recent significant developments identifying putative markers of these epithelial stem/progenitor cells that reveal their in vivo identity, location in both human and mouse endometrium, raising common but also different viewpoints. The review also outlines the techniques used to identify epithelial stem/progenitor cells, specifically in vitro functional assays and in vivo lineage tracing. We will also discuss their known interactions and hierarchy and known roles in endometrial dynamics across the menstrual or estrous cycle including re-epithelialization at menses and regeneration of the tissue during the proliferative phase. We also detail their potential role in endometrial proliferative disorders such as endometriosis.

Pharmacological Inhibition of the Stress-Related Deacetylase SIRT1 Enhances Eradication of CML stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 448-448
Author(s):  
Ling Li ◽  
Lisheng Wang ◽  
Liang Li ◽  
Tinisha McDonald ◽  
Yin Wei Ho ◽  
...  
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Blast Crisis ◽  
Fish Analysis ◽  
Pharmacological Inhibition ◽  
Knock Down ◽  
Cd34 Cells

Abstract Abstract 448 BCR-ABL tyrosine kinase inhibitors (TKI) are effective in inducing remissions and prolonging survival of CML patients, but fail to eradicate primitive leukemia stem cells (LSC) which remain a potential source of relapse. New strategies to enhance elimination of residual CML LSC in TKI-treated patients are required. We have previously reported that the stress-related deacetylase SIRT1 is expressed at high levels in CML stem/progenitor cells and that inhibition of SIRT1 expression using lentivirus-expressed shRNA induces apoptosis in CML progenitors and increases their sensitivity to imatinib (IM) by activating p53 signaling (Blood 2010, 116: 200A). These results support an important role for SIRT1 in CML LSC maintenance and TKI resistance, and as a potential molecular target for therapy directed against CML LSC. Tenovin-6 (TV) has been identified as a potent small molecule inhibitor of SIRT1 activity (Cancer Cell 2008, 13:454). Here we evaluated whether pharmacological inhibition of SIRT1 activity using TV could selectively inhibit CML stem/progenitor cells. As with shRNA-mediated knock-down of SIRT1, treatment with TV (0.5 μM) significantly increased apoptosis of CML CD34+ cells (TV16±7% vs. Control 3±2%, p=0.04, n=3), but not normal CD34+ cells (TV 6±2% vs. Control 4±2%, p=0.1, n=3). The combination of IM (2.5 μM) and TV induced significantly increased apoptosis in CML progenitors compared to IM alone, and to a significantly greater extent than in normal cells (CML, TV + IM 40±2% vs. IM 19±3%, p=0.009, n=3; CB, TV + IM 15±4% vs. IM 10±2%, p=0.04, n=3). TV (1 μM) increased apoptosis in both CML CD34+CD38− (TV 42±10% vs. Control 4±3%, p=0.04, n=3) and CD34+CD38+ cells (TV 35±7% vs. Control 8±2%, p=0.03, n=3). CFSE labeling indicated that treatment with TV resulted in increased apoptosis of undivided CML CD34+CD38− cells identified on the basis of high CFSE fluorescence (TV 20±7% vs. Control 2±1%, p=0.04, n=3). The combination of TV with IM resulted in a significant increase in apoptosis in CML CD34+CD38− CFSEhigh cells compared to IM alone (TV plus IM 35±5% vs. IM 10±4%, p=0.03, n=3). Treatment with TV (0.5 μM) reduced CML CFC frequency (70±9% inhibition with TV compared to untreated controls, p=0.009, n=3) without affecting normal CFC frequency. Combination of TV (0.5 μM) with IM resulted in enhanced inhibition of CML CFC compared to IM alone, but did not enhance inhibition of normal CFC (CML: TV plus IM 82±6% inhibition vs. IM 57±10%, p=0.02, n=3; CB: TV plus IM 38±7% inhibition vs. IM 36±9%, p=0.1, n=3). TV treatment effectively inhibited the growth of Baf3 cells expressing T315I-mutated BCR-ABL, and significantly enhanced apoptosis of IM-resistant CML blast crisis CD34+ cells [TV (1 μm) 30±1% vs. Control 19±5%, p=0.04, n=3], suggesting SIRT1 inhibition can also target TKI-resistant CML cells. Ex vivo treatment with TV (1 μM) significantly reduced longer-term (12 weeks) engraftment of CML CD34+ cells in NSG mice following TV treatment (TV treated 0.2*105±0.1*105 human CD45+ cells in murine BM vs. Control 1.8*105±0.6*105, p=0.009, n=5). Significant reduction in engraftment of CD33+ (p=0.008) and CD14+ myeloid cells (p=0.009) was seen. Q-PCR and FISH analysis confirmed that engrafted human cells were leukemic in origin. Interestingly, engraftment of CB CD34+ cells was not reduced after treatment with TV (TV 2.7*106±0.7*106 human CD45+ cells in murine BM, vs. Control 2.4*106±0.8*106, p=0.2, n=6). These results show that SIRT1 inhibition by TV effectively targets primitive human CML cells with in vivo multi-lineage engraftment capacity. Treatment with TV significantly enhanced acetylated p53 levels in CML CD34+ cells, indicating effective inhibition of SIRT1 activity. TV treatment also increased total p53 levels, possibly related to reduced p53 degradation. TV treatment did not increase acetylated p53 or total p53 levels in normal CD34+ cells. Importantly shRNA-mediated knock-down of p53 resulted in significant reduction of TV-induced apoptosis in CML CD34+ cells (13±6% apoptosis with p53 shRNA; 33±7% apoptosis with control shRNA, p=0.04, n=3), indicating that the effects of TV on CML CD34+ cells are related to p53 acetylation and activation. In conclusion, our studies indicate that pharmacological inhibition of SIRT1 can activate p53 and enhance eradication of CML LSC in combination with TKI treatment, and support further evaluation of targeted inhibition of SIRT1 as a therapeutic strategy in CML. Disclosures: No relevant conflicts of interest to declare.

Abstract 491: Injury-Activated TGFβ Recruits MSCs for Vascular Remodeling

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Mei Wan ◽  
Changjun Li ◽  
Gehua Zhen ◽  
Wenying He ◽  
Kai Jiao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Migration ◽  
Progenitor Cells ◽  
Vascular Remodeling ◽  
Ex Vivo ◽  
Vascular Lesions ◽  
Type I ◽  
Post Injury ◽  
Local Resident

Stem/progenitor cells can be recruited to participate in tissue remodeling, regeneration, or repair following injury. In particular, bone marrow-derived or local resident stem /progenitor cells are implicated in vascular repair and remodeling. It is believed that migratory factor(s) released by injured tissue creates a gradient to mediate the migration of stem cells to the injury sites. The key mediator(s) inducing the recruitment of the cells to the vascular lesions remains to be defined. Here we show that nestin + cells, which represent similar population as Sca1 + CD29 + CD11b - CD45 - mesenchymal/stromal stem cells (MSCs), were recruited and incorporated into the neointimal tissue in both rat model of balloon injury of carotid artery and mouse model of wire injury of femoral artery. Importantly, elevated active TGFβ1 level was observed in the injured vessels as early as 8 hr post injury, and the elevation lasted for 2 wks. To investigate whether injury-induced activation of TGFβ1 in vascular matrix stimulates migration of MSCs, we developed an aorta-conditioned medium (Aorta-CM)-based cell migration assay in which MSCs were placed in the upper chamber and the Aorta-CM prepared by culturing ex vivo injured aorta was placed in the lower chamber of a transwell chamber. Aorta-CM prepared from ex vivo injured aorta significantly enhanced cell migration compare to CM prepared from uninjured aorta. When neutralizing antibody specific for TGFβ1 or the inhibitor of TGFβ type I receptor (TβRI) was added to the CM, the migration of MSCs was almost abolished, indicating that active TGFβ1 released by the injured arteries mediates MSCs migration. To examine the role of TGFβ in MSCs recruitment following vascular injury in vivo , TβRI inhibitor was injected into mice with injury of femoral arteries. Both the development of neointima and the recruitment of nestin + cells were blocked with the inhibitor treatment compare to those with vehicle treatment. Collectively, the results suggest that TGFβ is an injury-activated messenger essential for the recruitment of MSCs to participate in vascular remodeling.

scholarly journals Electrically Induced Calcium Handling in Cardiac Progenitor Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Joshua T. Maxwell ◽  
Mary B. Wagner ◽  
Michael E. Davis
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Systematic Approach ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Calcium Handling ◽  
Cardiac Progenitor Cells ◽  
Intracellular Ca ◽  
Mechanistic Basis ◽  
And Function

For nearly a century, the heart was viewed as a terminally differentiated organ until the discovery of a resident population of cardiac stem cells known as cardiac progenitor cells (CPCs). It has been shown that the regenerative capacity of CPCs can be enhanced byex vivomodification. Preconditioning CPCs could provide drastic improvements in cardiac structure and function; however, a systematic approach to determining a mechanistic basis for these modifications founded on the physiology of CPCs is lacking. We have identified a novel property of CPCs to respond to electrical stimulation by initiating intracellular Ca2+oscillations. We used confocal microscopy and intracellular calcium imaging to determine the spatiotemporal properties of the Ca2+signal and the key proteins involved in this process using pharmacological inhibition and confocal Ca2+imaging. Our results provide valuable insights into mechanisms to enhance the therapeutic potential in stem cells and further our understanding of human CPC physiology.

scholarly journals In Vivo Assessment of Gene Delivery to Keratinocytes by Lentiviral Vectors

2002 ◽  
Vol 76 (3) ◽  
pp. 1496-1504 ◽  
Author(s):  
Ulrich Kuhn ◽  
Atsushi Terunuma ◽  
Wolfgang Pfutzner ◽  
Ruth Ann Foster ◽  
Jonathan C. Vogel
Keyword(s):  
Stem Cells ◽  
Progenitor Cells ◽  
Human Skin ◽  
Ex Vivo ◽  
Lentiviral Vectors ◽  
Retroviral Vectors ◽  
Immunocompromised Mice ◽  
In Vivo Assessment

ABSTRACT For skin gene therapy, introduction of a desired gene into keratinocyte progenitor or stem cells could overcome the problem of achieving persistent gene expression in a significant percentage of keratinocytes. Although keratinocyte stem cells have not yet been completely characterized and purified for gene targeting purposes, lentiviral vectors may be superior to retroviral vectors at gene introduction into these stem cells, which are believed to divide and cycle slowly. Our initial in vitro studies demonstrate that lentiviral vectors are able to efficiently transduce nondividing keratinocytes, unlike retroviral vectors, and do not require the lentiviral accessory genes for keratinocyte transduction. When lentiviral vectors expressing green fluorescent protein (GFP) were directly injected into the dermis of human skin grafted onto immunocompromised mice, transduction of dividing basal and nondividing suprabasal keratinocytes could be demonstrated, which was not the case when control retroviral vectors were used. However, flow cytometry analysis demonstrated low transduction efficiency, and histological analysis at later time points provided no evidence for progenitor cell targeting. In an alternative in vivo method, human keratinocytes were transduced in tissue culture (ex vivo) with either lentiviral or retroviral vectors and grafted as skin equivalents onto immunocompromised mice. GFP expression was analyzed in these human skin grafts after several cycles of epidermal turnover, and both the lentiviral and retroviral vector-transduced grafts had similar percentages of GFP-expressing keratinocytes. This ex vivo grafting study provides a good in vivo assessment of gene introduction into progenitor cells and suggests that lentiviral vectors are not necessarily superior to retroviral vectors at introducing genes into keratinocyte progenitor cells during in vitro culture.

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

2020 ◽  
Vol 15 ◽  
Author(s):  
Fatima Aerts-Kaya
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stress Conditions ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

scholarly journals Radiation-induced toxicity in rectal epithelial stem cell contributes to acute radiation injury in rectum

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Felipe Rodriguez Tirado ◽  
Payel Bhanja ◽  
Eduardo Castro-Nallar ◽  
Ximena Diaz Olea ◽  
Catalina Salamanca ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ex Vivo ◽  
Cre Recombinase ◽  
Lineage Tracing ◽  
Common Side Effect ◽  
Rectal Injury ◽  
Male Mice ◽  
Pelvic Irradiation ◽  
Pelvic Malignancies ◽  
Radiation Induced

Abstract Background Radiation-induced rectal epithelial damage is a very common side effect of pelvic radiotherapy and often compromise the life quality and treatment outcome in patients with pelvic malignancies. Unlike small bowel and colon, effect of radiation in rectal stem cells has not been explored extensively. Here we demonstrate that Lgr5-positive rectal stem cells are radiosensitive and organoid-based transplantation of rectal stem cells mitigates radiation damage in rectum. Methods C57Bl6 male mice (JAX) at 24 h were exposed to pelvic irradiation (PIR) to determine the radiation effect in pelvic epithelium. Effect of PIR on Lgr5-positive rectal stem cells (RSCs) was determined in Lgr5-EGFP-Cre-ERT2 mice exposed to PIR. Effect of PIR or clinically relevant fractionated PIR on regenerative response of Lgr5-positive RSCs was examined by lineage tracing assay using Lgr5-eGFP-IRES-CreERT2; Rosa26-CAG-tdTomato mice with tamoxifen administration to activate Cre recombinase and thereby marking the ISC and their respective progeny. Ex vivo three-dimensional organoid cultures were developed from Lgr5-EGFP-Cre-ERT2 mice. Organoid growth was determined by quantifying the budding crypt/total crypt ratio. Organoids from Lgr5-EGFP-ires-CreERT2-TdT mice were transplanted in C57Bl6 male mice exposed to PIR. Engraftment and repopulation of Lgr5-positive RSCs were determined after tamoxifen administration to activate Cre recombinase in recipient mice. Statistical analysis was performed using Log-rank (Mantel-Cox) test and paired two-tail t test. Result Exposure to pelvic irradiation significantly damaged rectal epithelium with the loss of Lgr5+ve rectal stem cells. Radiosensitivity of rectal epithelium was also observed with exposure to clinically relevant fractionated pelvic irradiation. Regenerative capacity of Lgr5+ve rectal stem cells was compromised in response to fractionated pelvic irradiation. Ex vivo organoid study demonstrated that Lgr5+ve rectal stem cells are sensitive to both single and fractionated radiation. Organoid-based transplantation of Lgr5+ve rectal stem cells promotes repair and regeneration of rectal epithelium. Conclusion Lgr5-positive rectal stem cells are radiosensitive and contribute to radiation-induced rectal epithelial toxicity. Transplantation of Lgr5-positive rectal stem cells mitigates radiation-induced rectal injury and promotes repair and regeneration process in rectum.

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