Somatic Stem Cell Maintenance Research Articles

PIWIL1 is recruited to centrosomes during mitosis in colorectal cancer cells and is linked to cell cycle progression

PIWI proteins, traditionally associated with germline development, have recently gained attention for their expression in various cancers, including colorectal cancer. However, the molecular mechanisms underlying their reactivation and impact on cancer initiation and progression remain elusive. Here, we found that PIWIL1 is expressed at relatively high levels in CRC-derived samples and cell lines, where it undergoes a dynamic relocalization to the centrosome during mitosis. Knockdown of PIWIL1 induces G2/M arrest associated with disruption of the mitotic spindle and aberrant metaphase events, highlighting its role in cell cycle progression. We also found that the expression of PIWIL1 is lost during the differentiation of Caco-2 cells into enterocytes and that PIWIL1 is expressed in cells at the base of the intestinal crypts in normal human colon tissue, where intestinal stem cells are known to reside. Thus, it is possible that the presence of PIWIL1 in cancer cells reflects a physiological role of this protein in stem cell maintenance, which would argue in favor of the proposed stem cell origin of CRC. Supporting this view, dedifferentiation of human fibroblasts into induced pluripotent stem cells (iPSCs) involves the reactivation of PIWIL2 expression, another member of the PIWI protein family. Overall, our findings suggest a role of PIWIL1 in mediating cell cycle dynamics, both in colorectal cancer cells and possibly also in intestinal stem cells. In a broader aspect, we provide evidence supporting an involvement of PIWI proteins in somatic stem cell maintenance, thus expanding the known non-gonadal functions of this protein family.

Escargot controls somatic stem cell maintenance through the attenuation of the insulin receptor pathway in Drosophila.

SummaryAdult stem cells coordinate intrinsic and extrinsic, local and systemic, cues to maintain the proper balance between self-renewal and differentiation. However, the precise mechanisms stem cells use to integrate these signals remain elusive. Here, we show that Escargot (Esg), a member of the Snail family of transcription factors, regulates the maintenance of somatic cyst stem cells (CySCs) in the Drosophila testis by attenuating the activity of the pro-differentiation insulin receptor (InR) pathway. Esg positively regulates the expression of an antagonist of insulin signaling, ImpL2, while also attenuating the expression of InR. Furthermore, Esg-mediated repression of the InR pathway is required to suppress CySC loss in response to starvation. Given the conservation of Snail-family transcription factors, characterizing the mechanisms by which Esg regulates cell-fate decisions during homeostasis and a decline in nutrient availability is likely to provide insight into the metabolic regulation of stem cell behavior in other tissues and organisms.

Hyperactivation of sympathetic nerves drives depletion of melanocyte stem cells.

SUMMARYEmpirical and anecdotal evidence have associated stress with accelerated hair greying (formation of unpigmented hairs)1,2, but the scientific evidence linking the two is scant. Here, we report that acute stress leads to hair greying through fast depletion of melanocyte stem cells (MeSCs). Combining adrenalectomy, denervation, chemogenetics3,4, cell ablation, and MeSC-specific adrenergic receptor knockout, we found that stress-induced MeSC loss is independent of immune attack or adrenal stress hormones. Rather, hair greying results from activation of the sympathetic nerves that innervate the MeSC niche. Upon stress, sympathetic nerve activation leads to burst release of the neurotransmitter norepinephrine, which drives quiescent MeSCs into rapid proliferation, followed by differentiation, migration, and permanent depletion from the niche. Transient suppression of MeSC proliferation prevents stress-induced hair greying. Our studies demonstrate that acute stress-induced neuronal activity can drive rapid and permanent loss of somatic stem cells, and illustrate an example in which somatic stem cell maintenance is directly influenced by the overall physiological state of the organism.

Loss of tyrosine kinase receptor Ephb2 impairs proliferation and stem cell activity of spermatogonia in culture†.

Germline stem and progenitor cells can be extracted from the adult mouse testis and maintained long-term in vitro. Yet, the optimal culture conditions for preserving stem cell activity are unknown. Recently, multiple members of the Eph receptor family were detected in murine spermatogonia, but their roles remain obscure. One such gene, Ephb2, is crucial for maintenance of somatic stem cells and was previously found enriched at the level of mRNA in murine spermatogonia. We detected Ephb2 mRNA and protein in primary adult spermatogonial cultures and hypothesized that Ephb2 plays a role in maintenance of stem cells in vitro. We employed CRISPR-Cas9 targeting and generated stable mutant SSC lines with complete loss of Ephb2. The characteristics of Ephb2-KO cells were interrogated using phenotypic and functional assays. Ephb2-KO SSCs exhibited reduced proliferation compared to wild-type cells, while apoptosis was unaffected. Therefore, we examined whether Ephb2 loss correlates with activity of canonical pathways involved in stem cell self-renewal and proliferation. Ephb2-KO cells had reduced ERK MAPK signaling. Using a lentiviral transgene, Ephb2 expression was rescued in Ephb2-KO cells, which partially restored signaling and proliferation. Transplantation analysis revealed that Ephb2-KO SSCs cultures formed significantly fewer colonies than WT, indicating a role for Ephb2 in preserving stem cell activity of cultured cells. Transcriptome analysis of wild-type and Ephb2-KO SSCs identified Dppa4 and Bnc1 as differentially expressed, Ephb2-dependent genes that are potentially involved in stem cell function. These data uncover for the first time a crucial role for Ephb2 signaling in cultured SSCs.

Wnt/β-Catenin Signaling: The Culprit in Pancreatic Carcinogenesis and Therapeutic Resistance.

Pancreatic ductal adenocarcinoma (PDAC) is responsible for 7.3% of all cancer deaths. Even though there is a steady increase in patient survival for most cancers over the decades, the patient survival rate for pancreatic cancer remains low with current therapeutic strategies. The Wnt/β-catenin pathway controls the maintenance of somatic stem cells in many tissues and organs and is implicated in pancreatic carcinogenesis by regulating cell cycle progression, apoptosis, epithelial-mesenchymal transition (EMT), angiogenesis, stemness, tumor immune microenvironment, etc. Further, dysregulated Wnt has been shown to cause drug resistance in pancreatic cancer. Although different Wnt antagonists are effective in pancreatic patients, limitations remain that must be overcome to increase the survival benefits associated with this emerging therapy. In this review, we have summarized the role of Wnt signaling in pancreatic cancer and suggested future directions to enhance the survival of pancreatic cancer patients.

MTORC1 Activation during Repeated Regeneration Impairs Somatic Stem Cell Maintenance

The balance between self-renewal and differentiation ensures long-term maintenance of stem cell (SC) pools in regenerating epithelial tissues. This balance is challenged during periods of high regenerative pressure and is often compromised in aged animals. Here, we show that target of rapamycin (TOR) signaling is a key regulator of SC loss during repeated regenerative episodes. In response to regenerative stimuli, SCs in the intestinal epithelium of the fly and in the tracheal epithelium of mice exhibit transient activation of TOR signaling. Although this activation is required for SCs to rapidly proliferate in response to damage, repeated rounds of damage lead to SC loss. Consistently, age-related SC loss in the mouse trachea and in muscle can be prevented by pharmacologic or genetic inhibition, respectively, of mammalian target of rapamycin complex 1 (mTORC1) signaling. These findings highlight an evolutionarily conserved role of TOR signaling in SC function and identify repeated rounds of mTORC1 activation as a driver of age-related SC decline.

CBP/Catenin antagonists: Targeting LSCs' Achilles heel.

Cancer stem cells (CSCs), including leukemia stem cells (LSCs), exhibit self-renewal capacity and differentiation potential and have the capacity to maintain or renew and propagate a tumor/leukemia. The initial isolation of CSCs/LSCs was in adult myelogenous leukemia, although more recently, the existence of CSCs in a wide variety of other cancers has been reported. CSCs, in general, and LSCs, specifically with respect to this review, are responsible for initiation of disease, therapeutic resistance and ultimately disease relapse. One key focus in cancer research over the past decade has been the development of therapies that safely eliminate the LSC/CSC population. One major obstacle to this goal is the identification of key mechanisms that distinguish LSCs from normal endogenous hematopoietic stem cells. An additional daunting feature that has recently come to light with advances in next-generation sequencing and single-cell sequencing is the heterogeneity within leukemias/tumors, with multiple combinations of mutations, gain and loss of function of genes, and so on being capable of driving disease, even within the CSC/LSC population. The focus of this review/perspective is on our work in identifying and validating, in both chronic myelogenous leukemia and acute lymphoblastic leukemia, a safe and efficacious mechanism to target an evolutionarily conserved signaling nexus, which constitutes a common "Achilles heel" for LSCs/CSCs, using small molecule-specific CBP/catenin antagonists.

Drosophila dyskerin is required for somatic stem cell homeostasis

Drosophila represents an excellent model to dissect the roles played by the evolutionary conserved family of eukaryotic dyskerins. These multifunctional proteins are involved in the formation of H/ACA snoRNP and telomerase complexes, both involved in essential cellular tasks. Since fly telomere integrity is guaranteed by a different mechanism, we used this organism to investigate the specific role played by dyskerin in somatic stem cell maintenance. To this aim, we focussed on Drosophila midgut, a hierarchically organized and well characterized model for stemness analysis. Surprisingly, the ubiquitous loss of the protein uniquely affects the formation of the larval stem cell niches, without altering other midgut cell types. The number of adult midgut precursor stem cells is dramatically reduced, and this effect is not caused by premature differentiation and is cell-autonomous. Moreover, a few dispersed precursors found in the depleted midguts can maintain stem identity and the ability to divide asymmetrically, nor show cell-growth defects or undergo apoptosis. Instead, their loss is mainly specifically dependent on defective amplification. These studies establish a strict link between dyskerin and somatic stem cell maintenance in a telomerase-lacking organism, indicating that loss of stemness can be regarded as a conserved, telomerase-independent effect of dyskerin dysfunction.

Mammary Stem Cell Self-Renewal Is Regulated by Slit2/Robo1 Signaling through SNAI1 and mINSC.

Tissue homeostasis requires somatic stem cell maintenance; however, mechanisms regulating this process during organogenesis are not well understood. Here, we identify asymmetrically renewing basal and luminal stem cells in the mammary end bud. We demonstrate that SLIT2/ROBO1 signaling regulates the choice between self-renewing asymmetric cell divisions (ACDs) and expansive symmetric cell divisions (SCDs) by governing Inscuteable (mInsc), a key member of the spindle orientation machinery, through the transcription factor Snail (SNAI1). Loss of SLIT2/ROBO1 signaling increases SNAI1 in the nucleus. Overexpression of SNAI1 increases mInsc expression, an effect that is inhibited by SLIT2 treatment. Increased mInsc does not change cell proliferation in the mammary gland (MG) but instead causes more basal cap cells to divide via SCD, at the expense of ACD, leading to more stem cells and larger outgrowths. Together, our studies provide insight into how the number of mammary stem cells is regulated by the extracellular cue SLIT2.

Non-neuronal functions of acetylcholine via muscarinic receptor subtypes: cellular proliferation and differentiation in mammals

Acetylcholine (ACh) is considered a neurotransmitter in central, parasympathetic, and neuromuscular synapses of mammals. Many of the important physiological functions of ACh are caused by the interaction of ACh with a group of G protein-coupled receptors (GPCRs) referred to as muscarinic ACh receptors (mAChRs). GPCRs are divided into five molecularly distinct muscarinic receptor subtypes that are referred to as M1-M5. M1-M5 receptors are abundantly expressed in most cells and tissues and are critically involved in cellular function, proliferation, and differentiation. Non-neuronal cells, including murine embryonic stem cells, epithelial cells, endothelial cells, and immune cells, synthesize ACh, which modulates cellular activities via ACh receptors (muscarinic and nicotinic receptors) in response to internal and external stimuli. Recently, we revealed that via activation of mAChRs, endogenously released ACh from intestinal epithelial cells contributes to the maintenance of somatic stem cells and the inhibition of differentiation to intestinal epithelial cells. Therefore, non-neuronal ACh production results in autocrine and paracrine promotion of cell proliferation and differentiation of embryonic and somatic stem cells.

Details Unfold: The Endoplasmic Reticulum Stress Response in Intestinal Inflammation and Cancer

Details Unfold: The Endoplasmic Reticulum Stress Response in Intestinal Inflammation and Cancer

Defining the genomic signature of the parous breast

BackgroundIt is accepted that a woman's lifetime risk of developing breast cancer after menopause is reduced by early full term pregnancy and multiparity. This phenomenon is thought to be associated with the development and differentiation of the breast during pregnancy.MethodsIn order to understand the underlying molecular mechanisms of pregnancy induced breast cancer protection, we profiled and compared the transcriptomes of normal breast tissue biopsies from 71 parous (P) and 42 nulliparous (NP) healthy postmenopausal women using Affymetrix Human Genome U133 Plus 2.0 arrays. To validate the results, we performed real time PCR and immunohistochemistry.ResultsWe identified 305 differentially expressed probesets (208 distinct genes). Of these, 267 probesets were up- and 38 down-regulated in parous breast samples; bioinformatics analysis using gene ontology enrichment revealed that up-regulated genes in the parous breast represented biological processes involving differentiation and development, anchoring of epithelial cells to the basement membrane, hemidesmosome and cell-substrate junction assembly, mRNA and RNA metabolic processes and RNA splicing machinery. The down-regulated genes represented biological processes that comprised cell proliferation, regulation of IGF-like growth factor receptor signaling, somatic stem cell maintenance, muscle cell differentiation and apoptosis.ConclusionsThis study suggests that the differentiation of the breast imprints a genomic signature that is centered in the mRNA processing reactome. These findings indicate that pregnancy may induce a safeguard mechanism at post-transcriptional level that maintains the fidelity of the transcriptional process.

Hh signalling is essential for somatic stem cell maintenance in the Drosophila testis niche

Hh signalling is essential for somatic stem cell maintenance in the <i>Drosophila</i> testis niche

Abstract 3672: Defining the genomic signature of the parous breast

Abstract Epidemiological data have consistently shown that a pregnancy early in life confer mothers lifetime protection against breast cancer, which is enhanced by subsequent pregnancies. Previous studies have demonstrated that the protection conferred by pregnancy is mediated by the differentiation of the breast, which is expressed as a specific genomic profile detectable in breast cells of postmenopausal parous women that serves as a biomarker indicative of reduced cancer risk. For further confirming these observations we designed a multidisciplinary study for determining whether the pattern of gene expression differed between nulliparous and parous postmenopausal women from an ethnically homogeneous population residing in Norrbotten County, Sweden; the study was approved by the IRB/Ethical Board of the University of Umeå, Sweden. Volunteer women from 50-69 years of age, belonging to one of the following categories based on reproductive history: parous (G≥1/P≥1), nulligravida nullipara (G0/P0) or gravida nullipara (G≥1/P0). The subjects signed an informed consent to participate in the study and donated core needle biopsies (CNB) of breast. CNB were taken from the upper outer quadrant of the breast and divided for 70% ethanol fixation for histopathological analysis and RNA extraction for subsequent genomic analysis. Total RNA was isolated using the Qiagen Allprep RNA/DNA Mini Kit. RNA quantity and quality were determined and the GeneChip Expression 3’-Amplification Two-Cycle cDNA Synthesis Kit (Affymetrix, Santa Clara, CA) was used to prepare the cRNA for hybridization; samples were hybridized to Affymetrix HG_U133 Plus 2.0 oligonucleotide arrays; 71 parous and 42 nulliparous satisfied quality control thresholds based on standard Affymetrix quality control measures. Probesets for which the proportion of Present calls was less than 75% in both samples were filtered out. A variance filter was also applied, removing all probesets whose variance across all samples fell below the first quartile, remaining 18,694 probesets for further analysis using both a p-value of 0.001 from the empirical Bayes moderated t-statistics, and a minimum log2 fold-change of 0.3 threshold as criteria of significance. Two hundred and eight genes were found to be differentially expressed between parous and nulliparous women. Gene ontology and pathway analyses revealed enrichment of biological processes related to regulation of transcription, RNA splicing, cell cycle control, adhesion and differentiation. IGF-like growth factor signaling and somatic stem cell maintenance were significantly downregulated. These results demonstrated that the breast of parous postmenopausal women exhibits a transcriptomic profile that differs from that of nulliparous women, representing a genomic signature induced by full term pregnancy that is indicative of breast differentiation. Work supported by Avon Foundation Women Breast Cancer Research Program grant 02-2008-034. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3672. doi:10.1158/1538-7445.AM2011-3672

The evolution of oocyte patterning in insects: multiple cell-signaling pathways are active during honeybee oogenesis and are likely to play a role in axis patterning

SUMMARY In Drosophila it is well established that signaling between the germline and surrounding follicle cells establishes the axes of the future embryo and is required for patterning of the eggshell. However, little is known about how this is achieved in other insects. Genome sequencing studies imply that maternal axis determination may be rapidly evolving, as a number of Drosophila maternal patterning genes are absent from the genomes of other insects. We have examined the distribution and function of six developmental signaling pathways present, and active, in honeybee ovarioles. We have confirmed an evolutionarily conserved role for transforming growth factor-α-epidermal growth factor receptor signaling in dorsal-ventral (DV) patterning. We also found evidence for the involvement of Dpp/Mad and JNK-MAPK pathways in DV patterning, unlike Drosophila. Several of these pathways are also active in the germarium, implicating them in germ and somatic stem cell maintenance and proliferation, similar to their activities in Drosophila ovaries.

G-protein Coupled Receptors in Stem Cell Self-Renewal and Differentiation

Stem cells have great potential for understanding early development, treating human disease, tissue trauma and early phase drug discovery. The factors that control the regulation of stem cell survival, proliferation, migration and differentiation are still emerging. Some evidence now exists demonstrating the potent effects of various G-protein coupled receptor (GPCR) ligands on the biology of stem cells. This review aims to give an overview of the current knowledge of the regulation of embryonic and somatic stem cell maintenance and differentiation by GPCR ligands.

Stem cells are differentially regulated during development, regeneration and homeostasis in flatworms

The flatworm stem cell system is exceptional within the animal kingdom, as totipotent stem cells (neoblasts) are the only dividing cells within the organism. In contrast to most organisms, piwi-like gene expression in flatworms is extended from germ cells to somatic stem cells. We describe the isolation and characterization of the piwi homologue macpiwi in the flatworm Macrostomum lignano. We use in situ hybridization, antibody staining and RNA interference to study macpiwi expression and function in adults, during postembryonic development, regeneration and upon starvation. We found novelties regarding piwi function and observed differences to current piwi functions in flatworms. First, macpiwi was essential for the maintenance of somatic stem cells in adult animals. A knock-down of macpiwi led to a complete elimination of stem cells and death of the animals. Second, the regulation of stem cells was different in adults and regenerates compared to postembryonic development. Third, sexual reproduction of M. lignano allowed to follow germline formation during postembryonic development, regeneration, and starvation. Fourth, piwi expression in hatchlings further supports an embryonic formation of the germline in M. lignano. Our findings address new questions in flatworm stem cell research and provide a basis for comparison with higher organisms.

Multipotent somatic stem cells contribute to the stem cell niche in the Drosophila testis

Adult stem cells reside in specialized microenvironments, or niches, that have an important role in regulating stem cell behaviour. Therefore, tight control of niche number, size and function is necessary to ensure the proper balance between stem cells and progenitor cells available for tissue homeostasis and wound repair. The stem cell niche in the Drosophila male gonad is located at the tip of the testis where germline and somatic stem cells surround the apical hub, a cluster of approximately 10-15 somatic cells that is required for stem cell self-renewal and maintenance. Here we show that somatic stem cells in the Drosophila testis contribute to both the apical hub and the somatic cyst cell lineage. The Drosophila orthologue of epithelial cadherin (DE-cadherin) is required for somatic stem cell maintenance and, consequently, the apical hub. Furthermore, our data indicate that the transcriptional repressor escargot regulates the ability of somatic cells to assume and/or maintain hub cell identity. These data highlight the dynamic relationship between stem cells and the niche and provide insight into genetic programmes that regulate niche size and function to support normal tissue homeostasis and organ regeneration throughout life.

Oct4 Expression Is Not Required for Mouse Somatic Stem Cell Self-Renewal

The Pou domain containing transcription factor Oct4 is a well-established regulator of pluripotency in the inner cell mass of the mammalian blastocyst as well as in embryonic stem cells. While it has been shown that the Oct4 gene is inactivated through a series of epigenetic modifications following implantation, recent studies have detected Oct4 activity in a variety of somatic stem cells and tumor cells. Based on these observations it has been suggested that Oct4 may also function in maintaining self-renewal of somatic stem cells and, in addition, may promote tumor formation. We employed a genetic approach to determine whether Oct4 is important for maintaining pluripotency in the stem cell compartments of several somatic tissues including the intestinal epithelium, bone marrow (hematopoietic and mesenchymal lineages), hair follicle, brain, and liver. Oct4 gene ablation in these tissues revealed no abnormalities in homeostasis or regenerative capacity. We conclude that Oct4 is dispensable for both self-renewal and maintenance of somatic stem cells in the adult mammal.

Cellular Senescence: Ex Vivo p53-Dependent Asymmetric Cell Kinetics.

Although senescence is a defining property of euploid mammalian cells, its physiologic basis remains obscure. Previously, cell kinetics properties of normal tissue cells have not been considered in models for senescence. We now provide evidence that senescence is in fact the natural consequence of normal in vivo somatic stem cell kinetics extended in culture. This concept of senescence is based on our discovery that cells engineered to conditionally express the well-recognized tumor suppressor protein and senescence factor, p53, exhibit asymmetric cell kinetics. In vivo, asymmetric cell kinetics are essential for maintenance of somatic stem cells; ex vivo, the same cell kinetics yield senescence as a simple kinetic endpoint. This new “asymmetric cell kinetics model” for senescence suggests novel strategies for the isolation and propagation of somatic tissue stem cells in culture.