Notch signaling regulates gastric antral LGR5 stem cell function

The major signaling pathways regulating gastric stem cells are unknown. Here we report that Notch signaling is essential for homeostasis of LGR5⁺ antral stem cells. Pathway inhibition reduced proliferation of gastric stem and progenitor cells, while activation increased proliferation. Notch dysregulation also altered differentiation, with inhibition inducing mucous and endocrine cell differentiation while activation reduced differentiation. Analysis of gastric organoids demonstrated that Notch signaling was intrinsic to the epithelium and regulated growth. Furthermore, in vivo Notch manipulation affected the efficiency of organoid initiation from glands and single Lgr5‐GFP stem cells, suggesting regulation of stem cell function. Strikingly, constitutive Notch activation in LGR5⁺ stem cells induced tissue expansion via antral gland fission. Lineage tracing using a multi‐colored reporter demonstrated that Notch‐activated stem cells rapidly generate monoclonal glands, suggesting a competitive advantage over unmanipulated stem cells. Notch activation was associated with increased mTOR signaling, and mTORC1 inhibition normalized NICD‐induced increases in proliferation and gland fission. Chronic Notch activation induced undifferentiated, hyper‐proliferative polyps, suggesting that aberrant activation of Notch in gastric stem cells may contribute to gastric tumorigenesis.     

Sex hormone drives blood stem cell reproduction

Stem cells ensure the maintenance of tissue homeostasis throughout life by tightly regulating their self‐renewal and differentiation. In a recent study published in Nature, Nakada et al, 2014 unveil an unexpected endocrine mechanism that regulates hematopoietic stem cell (HSC) self‐renewal.     

Ox‐LDL modifies the behaviour of bone marrow stem cells and impairs their endothelial differentiation via inhibition of Akt phosphorylation

This study was to investigate the effect of oxidized low‐density lipoprotein (ox‐LDL) on the behaviour of bone marrow stem cells and their endothelial differentiation as well as the underlying mechanisms. Adult rat bone marrow multipotent progenitor cells (MAPCs) were incubated with ox‐LDL for up to 2 weeks. Ox‐LDL treatment resulted in a time‐ and dose‐dependent reduction of MAPC population in culture through a combination of decreased cell proliferation and increased apoptosis. The expression of stem cell marker Oct‐4 was significantly suppressed in MAPCs by ox‐LDL in a dose‐ and time‐dependant manner. Endothelial differentiation of MAPCs was substantially inhibited by ox‐LDL with markedly decreased expression of endothelial markers vWF, Flk‐1 and CD31, as well as impaired in vitro vascular structure formation. Ox‐LDL‐induced apoptosis and inhibition of Oct‐4 expression, cell proliferation and endothelial differentiation of MAPCs were associated with significant inhibition of Akt phosphorylation. Akt overexpression in MAPCs transfected with a constitutively active Akt completely reversed the effects of ox‐LDL on MAPCs including enhanced apoptosis, decreased cell proliferation, suppressed Oct‐4 expression and endothelial differentiation as well as in vitro vascular structure formation. In conclusion, ox‐LDL promotes apoptosis and inhibits Oct‐4 expression and self‐renewal of MAPCs, and impairs their endothelial differentiation via suppression of Akt signalling.     

AMPKα1‐LDH pathway regulates muscle stem cell self‐renewal by controlling metabolic homeostasis

Control of stem cell fate to either enter terminal differentiation versus returning to quiescence (self‐renewal) is crucial for tissue repair. Here, we showed that AMP‐activated protein kinase (AMPK), the master metabolic regulator of the cell, controls muscle stem cell (MuSC) self‐renewal. AMPKα1^?/? MuSCs displayed a high self‐renewal rate, which impairs muscle regeneration. AMPKα1^?/? MuSCs showed a Warburg‐like switch of their metabolism to higher glycolysis. We identified lactate dehydrogenase (LDH) as a new functional target of AMPKα1. LDH, which is a non‐limiting enzyme of glycolysis in differentiated cells, was tightly regulated in stem cells. In functional experiments, LDH overexpression phenocopied AMPKα1^?/? phenotype, that is shifted MuSC metabolism toward glycolysis triggering their return to quiescence, while inhibition of LDH activity rescued AMPKα1^?/? MuSC self‐renewal. Finally, providing specific nutrients (galactose/glucose) to MuSCs directly controlled their fate through the AMPKα1/LDH pathway, emphasizing the importance of metabolism in stem cell fate.     

Aneuploid embryonic stem cells exhibit impaired differentiation and increased neoplastic potential

Aneuploidy leads to severe developmental defects in mammals and is also a hallmark of cancer. However, whether aneuploidy is a driving cause or a consequence of tumor formation remains controversial. Paradoxically, existing studies based on aneuploid yeast and mouse fibroblasts have shown that aneuploidy is usually detrimental to cellular fitness. Here, we examined the effects of aneuploidy on mouse embryonic stem (ES) cells by generating a series of cell lines that each carries an extra copy of single chromosomes, including trisomy 6, 8, 11, 12, or 15. Most of these aneuploid cell lines had rapid proliferation rates and enhanced colony formation efficiencies. They were less dependent on growth factors for self‐renewal and showed a reduced capacity to differentiate in vitro. Moreover, trisomic stem cells formed teratomas more efficiently, from which undifferentiated cells can be recovered. Further investigations demonstrated that co‐culture of wild‐type and aneuploid ES cells or supplementation with extracellular BMP4 rescues the differentiation defects of aneuploid ES cells.     

Notch signalling inhibits the adipogenic differentiation of single‐cell‐derived mesenchymal stem cell clones isolated from human adipose tissue

ADSCs (adipose‐derived mesenchymal stem cells) are candidate adult stem cells for regenerative medicine. Notch signalling participates in the differentiation of a heterogeneous ADSC population. We have isolated, human adipose tissue‐derived single‐cell clones using a cloning ring technique and characterized for their stem cell characteristics. The role of Notch signalling in the differentiation capacity of these adipose‐derived single‐cell‐clones has also been investigated. All 14 clones expressed embryonic and mesenchymal stem cell marker genes. These clones could differentiate into both osteogenic and adipogenic lineages. However, the differentiation potential of each clone was different. Low adipogenic clones had significantly higher mRNA expression levels of Notch 2, 3 and 4, Jagged1, as well as Delta1, compared with those of high adipogenic clones. In contrast, no changes in expression of Notch signalling component mRNA between low and high osteogenic clones was found. Notch receptor mRNA expression decreased with the adipogenic differentiation of both low and high adipogenic clones. The γ‐secretase inhibitor, DAPT (N‐[N‐(3,5‐difluorophenacetyl)‐l ‐alanyl]‐(S)‐phenylglycine t‐butyl ester), enhanced adipogenic differentiation. Correspondingly, cells seeded on a Notch ligand (Jagged1) bound surface showed lower intracellular lipid accumulation. These results were noted in both low and high adipogenic clones, indicating that Notch signalling inhibited the adipogenic differentiation of adipose ADSC clones, and could be used to identify an adipogenic susceptible subpopulation for soft‐tissue augmentation application.     

Rho‐kinase inhibitor Y‐27632 facilitates the proliferation,migration and pluripotency of human periodontal ligament stem cells

The selective in vitro expansion and differentiation of multipotent stem cells are critical steps in cell‐based regenerative therapies, while technical challenges have limited cell yield and thus affected the success of these potential treatments. The Rho GTPases and downstream Rho kinases are central regulators of cytoskeletal dynamics during cell cycle and determine the balance between stem cells self‐renewal, lineage commitment and apoptosis. Trans‐4‐[(1R)‐aminoethyl]‐N‐(4‐pyridinyl)cylohexanecarboxamidedihydrochloride (Y‐27632), Rho‐associated kinase (ROCK) inhibitor, involves various cellular functions that include actin cytoskeleton organization, cell adhesion, cell motility and anti‐apoptosis. Here, human periodontal ligament stem cells (PDLSCs) were isolated by limiting dilution method. Cell counting kit‐8 (CCK8), 5‐ethynyl‐2′‐deoxyuridine (EdU) labelling assay, cell apoptosis assay, cell migration assay, wound‐healing assay, alkaline phosphatase (ALP) activity assay, Alizarin Red S staining, Oil Red O staining, quantitative real‐time polymerase chain reaction (qRT‐PCR) were used to determine the effects of Y‐27632 on the proliferation, apoptosis, migration, stemness, osteogenic and adipogenic differentiation of PDLSCs. Afterwards, Western blot analysis was performed to elucidate the mechanism of cell proliferation. The results indicated that Y‐27632 significantly promoted cell proliferation, chemotaxis, wound healing, fat droplets formation and pluripotency, while inhibited ALP activity and mineral deposition. Furthermore, Y‐27632 induced PDLSCs proliferation through extracellular‐signal‐regulated kinase (ERK) signalling cascade. Therefore, control of Rho‐kinase activity may enhance the efficiency of stem cell‐based treatments for periodontal diseases and the strategy may have the potential to promote periodontal tissue regeneration by facilitating the chemotaxis of PDLSCs to the injured site, and then enhancing the proliferation of these cells and maintaining their pluripotency.     

Notch signaling regulates neuroepithelial stem cell maintenance and neuroblast formation in Drosophila optic lobe development

Notch signaling mediates multiple developmental decisions in Drosophila. In this study, we have examined the role of Notch signaling in Drosophila larval optic lobe development. Loss of function in Notch or its ligand Delta leads to loss of the lamina and a smaller medulla. The neuroepithelial cells in the optic lobe in Notch or Delta mutant brains do not expand but instead differentiate prematurely into medulla neuroblasts, which lead to premature neurogenesis in the medulla. Clonal analyses of loss-of-function alleles for the pathway components, including N, Dl, Su(H), and E(spl)-C, indicate that the Delta/Notch/Su(H) pathway is required for both maintaining the neuroepithelial stem cells and inhibiting medulla neuroblast formation while E(spl)-C is only required for some aspects of the inhibition of medulla neuroblast formation. Conversely, Notch pathway overactivation promotes neuroepithelial cell expansion while suppressing medulla neuroblast formation and neurogenesis; numb loss of function mimics Notch overactivation, suggesting that Numb may inhibit Notch signaling activity in the optic lobe neuroepithelial cells. Thus, our results show that Notch signaling plays a dual role in optic lobe development, by maintaining the neuroepithelial stem cells and promoting their expansion while inhibiting their differentiation into medulla neuroblasts. These roles of Notch signaling are strikingly similar to those of the JAK/STAT pathway in optic lobe development, raising the possibility that these pathways may collaborate to control neuroepithelial stem cell maintenance and expansion, and their differentiation into the progenitor cells.     

Critical roles of hMAGEA2 in induced pluripotent stem cell pluripotency,proliferation, and differentiation

Induced pluripotent stem (iPS) cells are important for clinical application and stem cell research. Although human melanoma‐associated antigen A2 (hMAGEA2) expression is known to affect differentiation in embryonic stem cells, its specific role in iPS cells remains unclear. To evaluate the function of hMAGEA2 and its characteristics in iPS cells, we produced hMAGEA2‐overexpressing iPS cells from hMAGEA2‐overexpressing transgenic mice. Although the iPS cells with overexpressed hMAGEA2 did not differ in morphology, their pluripotency, and self‐renewal related genes (Nanog, Oct3/4, Sox2, and Stat3), expression level was significantly upregulated. Moreover, hMAGEA2 contributed to the promotion of cell cycle progression, thereby accelerating cell proliferation. Through embryoid body formation in vitro and teratoma formation in vivo, we demonstrated that hMAGEA2 critically decreases the differentiation ability of iPS cells. These data indicate that hMAGEA2 intensifies the self‐renewal, pluripotency, and degree of proliferation of iPS cells, while significantly repressing their differentiation efficiency. Therefore, our findings prove that hMAGEA2 plays key roles in iPS cells.     

Using embryonic stem cells to form a biological pacemaker via tissue engineering technology

Biological pacemakers can be achieved by various gene‐based and cell‐based approaches. Embryonic stem cells (ESCs)‐derived pacemaker cells might be the most promising way to form biological pacemakers, but there are challenges as to how to control the differentiation of ESCs and to overcome the neoplasia, proarrhythmia, or immunogenicity resulting from the use of ESCs. As a potential approach to solve these difficult problems, tissue‐engineering techniques may provide a precise control on the different cell components of multicellular aggregates and the forming of a construct with‐defined architectures and functional properties. The combined interactions between ESC‐derived pacemaker cells, supporting cells, and matrices may completely reproduce pacemaker properties and result in a steady functional unit to induce rhythmic electrical and contractile activities. As ESCs have a high capability for self‐renewal, proliferation, and potential differentiation, we hypothesize that ESCs can be used as a source of pacemaker cells for tissue‐engineering applications and the ambitious goal of biological cardiac pacemakers may ultimately be achieved with ESCs via tissue‐engineering technology.     

Biophysical factors in the regulation of asymmetric division of stem cells

Stem cells are a promising cell source for regenerative medicine due to their characteristics of self‐renewal and differentiation. The intricate balance between these two cell fates is maintained by precisely controlled symmetric and asymmetric cell divisions. Asymmetric division has a fundamental importance in maintaining tissue homeostasis and in the development of multi‐cellular organisms. For example, during development, asymmetric cell divisions are responsible for the formation of the body axis. Mechanistically, mitotic spindle dynamics determine the assembly and separation of chromosomes and regulate the orientation of cell division. Interestingly, symmetric and asymmetric cell division is not mutually exclusive and a range of factors are involved in such cell‐fate decisions, the measurement of which can provide efficient and reliable information on the regenerative potential of a cell. The balance between self‐renewal and differentiation in stem cells is controlled by various biophysical and biochemical cues. Although the role of biochemical factors in asymmetric stem cell division has been widely studied, the effect of biophysical cues in stem‐cell self‐renewal is not comprehensively understood. Herein, we review the biological relevance of stem‐cell asymmetric division to regenerative medicine and discuss the influences of various intrinsic and extrinsic biophysical cues in stem‐cell self‐renewal. This review particularly aims to inform the clinical translation of efforts to control the self‐renewal ability of stem cells through the tuning of various biophysical cues.     

Lin28a enhances in vitro osteoblastic differentiation of human periosteum‐derived cells

Despite a capacity for proliferation and an ability to differentiate into multiple cell types, in long‐term culture and with ageing, stem cells show a reduction in growth, display a decrease in differentiation potential, and enter senescence without evidence of transformation. The Lin28a gene encodes an RNA‐binding protein that plays a role in regulating stem cell activity, including self‐renewal and differentiation propensity. However, the effect of the Lin28a gene on cultured human osteoprecursor cells is poorly understood. In the present study, alkaline phosphatase activity, alizarin red‐positive mineralization, and calcium content, positive indicators of osteogenic differentiation, were significantly higher in cultured human periosteum‐derived cells (hPDCs) with Lin28a overexpression compared with cells without Lin28a overexpression. Lin28a overexpression by hPDCs also increased mitochondrial activity, which is essential for cellular proliferation, as suggested by a reduced presence of reactive oxygen species and significantly enhanced lactate levels and ATP production. Our results suggest that, in hPDCs, the Lin28a gene enhances osteoblastic differentiation and increases mitochondrial activity. Although Lin28a is known as a marker of undifferentiated human embryogenic stem cell, there is limited evidence regarding the influence of Lin28a on osteoblastic differentiation of cultured osteoprecursor cells. This study was to examine the impact of Lin28a on osteogenic phenotypes of human periosteum‐derived cells. Their phenotypes can be similar to those of mesenchymal stem cells. Our results suggest that the Lin28a gene enhances the osteoblastic differentiation of human periosteum‐derived cells. In addition, the Lin28a gene increases mitochondrial activity in human periosteum‐derived cells.     

Depletion of HOXA5 inhibits the osteogenic differentiation and proliferation potential of stem cells from the apical papilla

Mesenchymal stem cells (MSCs) are a prospective cell source for tissue regeneration due to their self‐renewal abilities and potential to differentiate into different cell lineages, but the molecular mechanisms of the directed differentiation and proliferation are still unknown. Recently, multiple studies have indicated the crucial role of HOX genes in MSC differentiation and proliferation. However, the role of HOXA5 in MSCs remains unknown. Here, we investigated HOXA5 function in stem cells from the apical papilla (SCAPs). After HOXA5 depletion, the results showed a significant decrease in ALP activity and a weakened mineralization ability of SCAPs. The real‐time RT‐PCR results showed prominently lessened expression of OPN and BSP. The CCK8 and CFSE results displayed inhibited proliferation of SCAPs, and flow cytometry assays revealed arrested cell cycle progression at the S phase. Furthermore, we found that depletion of HOXA5 upregulated p16^INK4A and p18^INK4C and downregulated the Cyclin A. Our research demonstrated that depletion of HOXA5 inhibited osteogenic differentiation and repressed cell proliferation by arresting cell cycle progression at the S phase via p16^INK4A, p18^INK4C, and Cyclin A in SCAPs, indicating that HOXA5 has a significant role in maintaining the proliferation and differentiation potential of dental‐tissue‐derived MSCs.     

Identification of single chain antibodies to breast cancer stem cells using phage display

Recent evidence suggests that most malignancies are driven by “cancer stem cells” sharing the signature characteristics of adult stem cells: the ability to self renew and to differentiate. Furthermore these cells are thought to be quiescent, infrequently dividing cells with a natural resistance to chemotherapeutic agents. These studies theorize that therapies, which effectively treat the majority of tumor cells but ‘miss’ the stem cell population, will fail, while therapies directed at stern cells can potentially eradicate tumors. In breast cancer, researchers have isolated ‘breast cancer stem cells’ capable of recreating the tumor in vivo and in vitro. Generated new tumors contained both additional numbers of cancer stem cells and diverse mixed populations of cells present in the initial tumor, supporting the intriguing self‐renewal and differentiation characteristics. In the present study, an antibody phage library has been used to search for phage displayed‐single chain antibodies (scFv) with selective affinity to specific targets on breast cancer stem cells. We demonstrate evidence of two clones binding specifically to a cancer stem cell population isolated from the SUMl59 breast cancer cell line. These clones had selective affinity for cancer stem cells and they were able to select cancer stem cells among a large population of non‐stem cancer cells in paraffin‐embedded sections. The applicability of these clones to paraffin sections and frozen tissue specimens made them good candidates to be used as diagnostic and prognostic markers in breast cancer patient samples taking into consideration the cancer stern cell concept in tumor biology. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Cyclin D2 and the CDK substrate p220NPAT are required for self‐renewal of human embryonic stem cells

Self‐renewal of pluripotent human embryonic stem (hES) cells utilizes an abbreviated cell cycle that bypasses E2F/pRB‐dependent growth control. We investigated whether self‐renewal is alternatively regulated by cyclin/CDK phosphorylation of the p220^NPAT/HiNF‐P complex to activate histone gene expression at the G1/S phase transition. We show that cyclin D2 is prominently expressed in pluripotent hES cells, but cyclin D1 eclipses cyclin D2 during differentiation. Depletion of cyclin D2 or p220^NPAT causes a cell cycle defect in G1 reflected by diminished phosphorylation of p220^NPAT, decreased cell cycle dependent histone H4 expression and reduced S phase progression. Thus, cyclin D2 and p220^NPAT are principal cell cycle regulators that determine competency for self‐renewal in pluripotent hES cells. While pRB/E2F checkpoint control is relinquished in human ES cells, fidelity of physiological regulation is secured by cyclin D2 dependent activation of the p220^NPAT/HiNF‐P mechanism that may explain perpetual proliferation of hES cells without transformation or tumorigenesis. J. Cell. Physiol. 222: 456–464, 2010. © 2009 Wiley‐Liss, Inc.     

Smurf2 regulates hematopoietic stem cell self‐renewal and aging

The age‐dependent decline in the self‐renewal capacity of stem cells plays a critical role in aging, but the precise mechanisms underlying this decline are not well understood. By limiting proliferative capacity, senescence is thought to play an important role in age‐dependent decline of stem cell self‐renewal, although direct evidence supporting this hypothesis is largely lacking. We have previously identified the E3 ubiquitin ligase Smurf2 as a critical regulator of senescence. In this study, we found that mice deficient in Smurf2 had an expanded hematopoietic stem cell (HSC) compartment in bone marrow under normal homeostatic conditions, and this expansion was associated with enhanced proliferation and reduced quiescence of HSCs. Surprisingly, increased cycling and reduced quiescence of HSCs in Smurf2‐deficient mice did not lead to premature exhaustion of stem cells. Instead, HSCs in aged Smurf2‐deficient mice had a significantly better repopulating capacity than aged wild‐type HSCs, suggesting that decline in HSC function with age is Smurf2 dependent. Furthermore, Smurf2‐deficient HSCs exhibited elevated long‐term self‐renewal capacity and diminished exhaustion in serial transplantation. As we found that the expression of Smurf2 was increased with age and in response to regenerative stress during serial transplantation, our findings suggest that Smurf2 plays an important role in regulating HSC self‐renewal and aging.