Regulation of intestinal stem cells in mammals and Drosophila

The digestive systems in mammals and Drosophila are quite different in terms of their complexity and organization, but their biological functions are similar. The Drosophila midgut is a functional equivalent of the mouse small intestine. Adult intestinal stem cells (ISCs) have been identified in both the mouse small intestine and Drosophila midgut. The anatomy and cell renewal in the Drosophila midgut are similar to those in the mouse small intestine: the intestinal epithelium in both systems is a tube composed of epithelial cells with absorptive and secretory functions; the Notch signaling controls absorptive versus secretory fate decisions in the intestinal epithelium; cell renewal in both systems starts from stem cells in the basal cell layer, and the differentiated cells then move toward the lumen. However, it is clear that the stem cells in the two systems are regulated in different ways. In this review, we will compare cell renewal and stem cell regulation in the two systems. J. Cell. Physiol. 222:33–37, 2010. © 2009 Wiley‐Liss, Inc.     

Tumor suppressors Sav/scrib and oncogene ras regulate stem‐cell transformation in adult Drosophila malpighian tubules

An increasing body of evidence suggests that tumors might originate from a few transformed cells that share many properties with normal stem cells. However, it remains unclear how normal stem cells are transformed into cancer stem cells (CSCs). Here, we demonstrated that mutations causing the loss of tumor suppressor Salvador (Sav) or Scribble (Scrib) or activation of the oncogene Ras transform normal stem cells into CSCs through a multistep process in the adult Drosophila Malpighian Tubules (MTs). In wild‐type MTs, each stem cell generates one self‐renewing and one differentiating daughter cell. However, in flies with loss‐of‐function sav or scrib or gain‐of‐function Ras mutations, both daughter cells grew and behaved like stem cells, leading to the formation of tumors in MTs. Ras functioned downstream of Sav and Scrib in regulating the stem‐cell transformation. The Ras‐transformed stem cells exhibited many of the hallmarks of cancer, such as increased proliferation, reduced cell death, and failure to differentiate. We further demonstrated that several signal transduction pathways (including MEK/MAPK, RhoA, PKA, and TOR) mediate Ras' function in the stem‐cell transformation. Therefore, we have identified a molecular mechanism that regulates stem‐cell transformation, and this finding may lead to strategies for preventing tumor formation in certain organs. J. Cell. Physiol. 224: 766–774, 2010. © 2010 Wiley‐Liss, Inc.     

Induction of primordial germ cells from mouse induced pluripotent stem cells derived from adult hepatocytes

Pluripotent stem cells can be established by various methods, but they share several cytological properties, including germ cell differentiation in vitro, independently of their origin. Although mouse induced pluripotent stem (iPS) cells can produce functional gametes in vivo, it is still unclear whether or not they have the ability to produce presumptive germ cells in vitro. Here, we show that mouse iPS cells derived from adult hepatocytes were able to differentiate into presumptive germ cells marked by mouse vasa homolog (Mvh) expression in feeder‐free or suspension cultures. Embryoid body (EB) formation from iPS cells also induced the formation of round‐shaped cells resembling immature oocytes. Mvh⁺ cells formed clumps by co‐aggregation with differentiation‐supporting cells, and increased expression of germ cell markers was detected in these cell aggregates. Differentiation culture of presumptive germ cells from iPS cells could provide a conventional system for facilitating our understanding of the mechanisms underlying direct reprogramming and germline competency. Mol. Reprod. Dev. 77: 802–811, 2010. © 2010 Wiley‐Liss, Inc.     

magu is required for germline stem cell self-renewal through BMP signaling in the Drosophila testis

Understanding how stem cells are maintained in their microenvironment (the niche) is vital for their application in regenerative medicine. Studies of Drosophila male germline stem cells (GSCs) have served as a paradigm in niche-stem cell biology. It is known that the BMP and JAK-STAT pathways are necessary for the maintenance of GSCs in the testis (Kawase et al., 2004; Kiger et al., 2001; Schulz et al., 2004; Shivdasani and Ingham, 2003; Tulina and Matunis, 2001). However, our recent work strongly suggests that BMP signaling is the primary pathway leading to GSC self-renewal (Leatherman and DiNardo, 2010). Here we show that magu controls GSC maintenance by modulating the BMP pathway. We found that magu was specifically expressed from hub cells, and accumulated at the testis tip. Testes from magu mutants exhibited a reduced number of GSCs, yet maintained a normal population of somatic stem cells and hub cells. Additionally, BMP pathway activity was reduced, whereas JAK-STAT activation was retained in mutant testes. Finally, GSC loss caused by the magu mutation could be suppressed by overactivating the BMP pathway in the germline.     

E(nos)/CG4699 required for nanos function in the female germ line of Drosophila

The translational repressor Nanos is required in the germ line stem cells of the Drosophila ovary to maintain their capacity for self‐renewal. Following division of the stem cells, Nanos is inhibited in the daughters that differentiate into cysts and ultimately become mature oocytes. The control of Nanos activity is thus an important aspect of the switch from self‐renewal to differentiation. In this report, we describe a genetic interaction between nanos and Enhancer of nos, an allele of the previously uncharacterized locus CG4699. We find that E(nos) protein is required for normal accumulation of Nanos in the ovary and thus for maintenance of the germ line. The mechanism by which E(nos)/CG4699 protein acts is not clear, although it has been found in a complex with Mof acetylase. Consistent with the finding that E(nos) interacts with Mof, we observe that nanos and mof also interact genetically to maintain normal oogenesis. genesis 48:161–170, 2010. © 2010 Wiley‐Liss, Inc.     

Pluripotent plasticity of stem cells and liver repopulation

Different types of stem cells have a role in liver regeneration or fibrous repair during and after several liver diseases. Otherwise, the origin of hepatic and/or extra‐hepatic stem cells in reactive liver repopulation is under controversy. The ability of the human body to self‐repair and replace the cells and tissues of some organs is often evident. It has been estimated that complete renewal of liver tissue takes place in about a year. Replacement of lost liver tissues is accomplished by proliferation of mature hepatocytes, hepatic oval stem cells differentiation, and sinusoidal cells as support. Hepatic oval cells display a distinct phenotype and have been shown to be a bipotential progenitor of two types of epithelial cells found in the liver, hepatocytes, and bile ductular cells. In gastroenterology and hepatology, the first attempts to translate stem cell basic research into novel therapeutic strategies have been made for the treatment of several disorders, such as inflammatory bowel diseases, diabetes mellitus, celiachy, and acute or chronic hepatopaties. In the future, pluripotent plasticity of stem cells will open a variety of clinical application strategies for the treatment of tissue injuries, degenerated organs. The promise of liver stem cells lie in their potential to provide a continuous and readily available source of liver cells that can be used for gene therapy, cell transplant, bio‐artificial liver‐assisted devices, drug toxicology testing, and use as an in vitro model to understand the developmental biology of the liver. Copyright © 2010 John Wiley & Sons, Ltd.     

Female germline stem cell niches of earwigs are structurally simple and different from those of Drosophila melanogaster

Stem cells function in niches, which consist of somatic cells that control the stem cells' self‐renewal, proliferation, and differentiation. Drosophila ovary germline niche consists of the terminal filament (TF) cells, cap cells, and escort stem cells; signaling from the TF cells and the cap cells is essential for maintenance of germline stem cells (GSCs). Here, we show that in the earwig Opisthocosmia silvestris, the female GSC niche is morphologically simple and consist of the TF cells and several structurally uniform escort cells. The most posterior cell of the TF (the basal cell of the TF) differs from remaining TF cells and is separated from the anterior region of the germarium by the processes of the escort cells, and consequently, does not contact the GSCs directly. We also show that between somatic cells of earwig niche argosome‐like vesicles and cytoneme‐like extensions are present. J. Morphol., 2010. © 2009 Wiley‐Liss, Inc.     

Recreating the perivascular niche ex vivo using a microfluidic approach

Stem cell niches are composed of numerous microenvironmental features, including soluble and insoluble factors, cues from other cells, and the extracellular matrix (ECM), which collectively serve to maintain stem cell quiescence and promote their ability to support tissue homeostasis. A hallmark of many adult stem cell niches is their proximity to the vasculature in vivo, a feature common to neural stem cells, mesenchymal stem cells (MSCs) from bone marrow and adipose tissue, hematopoietic stem cells, and many tumor stem cells. In this study, we describe a novel 3D microfluidic device (MFD) as a model system in which to study the molecular regulation of perivascular stem cell niches. Endothelial cells (ECs) suspended within 3D fibrin gels patterned in the device adjacent to stromal cells (either fibroblasts or bone marrow‐derived MSCs) executed a morphogenetic process akin to vasculogenesis, forming a primitive vascular plexus and maturing into a robust capillary network with hollow well‐defined lumens. Both MSCs and fibroblasts formed pericytic associations with the ECs but promoted capillary morphogenesis with distinct kinetics. Biochemical assays within the niche revealed that the perivascular association of MSCs required interaction between their α6β1 integrin receptor and EC‐deposited laminin. These studies demonstrate the potential of this physiologically relevant ex vivo model system to study how proximity to blood vessels may influence stem cell multipotency. Biotechnol. Bioeng. 2010;107: 1020–1028. © 2010 Wiley Periodicals, Inc.     

Epithelial stem cells

New molecular markers for epidermal stem cells have enabled their isolation both in vitro and from the epidermis lying between hair follicles. Micro-dissection experiments have localised a second population of stem cells within hair follicles. Epidermal stem cells have a patterned distribution in vivo. The patterning can be reconstituted in vitro, showing that it is generated by interactions between keratinocytes and that the differentiation of epidermal stem cells is regulated by signals from other keratinocytes. Recent evidence from transgenic mice suggests that stem cell behaviour in the gut may be regulated by similar cell-cell interactions in vivo. Candidate genes for mediating these interactions are the homologues of Drosophila cell fate patterning genes such as Notch and Wingless and the Cadherin family of cell-cell adhesion molecules. The roles of stem cells and of mutations of the Patched gene in epithelial carcinogenesis are discussed.     

Stem cell potential for type 1 diabetes therapy

Stem cells have been considered as a useful tool in Regenerative Medicine due to two main properties: high rate of self-renewal, and their potential to differentiate into all cell types present in the adult organism. Depending on their origin, these cells can be grouped into embryonic or adult stem cells. Embryonic stem cells are obtained from the inner cell mass of blastocyst, which appears during embryonic day 6 of human development. Adult stem cells are present within various tissues of the organism and are responsible for their turnover and repair. In this sense, these cells open new therapeutic possibilities to treat degenerative diseases such as type 1 diabetes. This pathology is caused by the autoimmune destruction of pancreatic β-cells, resulting in the lack of insulin production. Insulin injection, however, cannot mimic β-cell function, thus causing the development of important complications. The possibility of obtaining β-cell surrogates from either embryonic or adult stem cells to restore insulin secretion will be discussed in this review.     

Cancer hallmarks in induced pluripotent cells: New insights

Studies are beginning to emerge that demonstrate intriguing differences between human‐induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). Here, we investigated the expression of key members of the Nodal embryonic signaling pathway, critical to the maintenance of pluripotency in hESCs. Western blot and real‐time RT‐PCR analyses reveal slightly lower levels of Nodal (a TGF‐β family member) and Cripto‐1 (Nodal's co‐receptor) and a dramatic decrease in Lefty (Nodal's inhibitor and TGF‐β family member) in hiPSCs compared with hESCs. The noteworthy drop in hiPSC's Lefty expression correlated with an increase in the methylation of Lefty B CpG island. Based on these findings, we addressed a more fundamental question related to the consequences of epigenetically reprogramming hiPSCs, especially with respect to maintaining a stable ESC phenotype. A global comparative analysis of 365 microRNAs (miRs) in two hiPSC versus four hESC lines ultimately identified 10 highly expressed miRs in hiPCSs with >10‐fold difference, which have been shown to be cancer related. These data demonstrate cancer hallmarks expressed by hiPSCs, which will require further assessment for their impact on future therapies. J. Cell. Physiol. 225: 390–393, 2010. © 2010 Wiley‐Liss, Inc.     

Optimization of immunomagnetic separation for cord blood-derived hematopoietic stem cells

Background  

There is a growing interest in cord blood as a source of primitive stem cells with the capacity for multilineage differentiation. Pure cell fractions are needed for the characterization and in vitro expansion of stem cells as well as for their use in preclinical research. However, enrichment of stem cells is challenging due to the lack of stem cell-specific markers and gentle protocols for the isolation of highly pure stem cell fractions. Protocols developed for the enrichment of peripheral blood-derived stem cells have been found to be suboptimal for cord blood.     

Regulation of active and quiescent somatic stem cells by Notch signaling

Somatic stem/progenitor cells actively proliferate and give rise to different types of mature cells (active state) in embryonic tissues while they are mostly dormant (quiescent state) in many adult tissues. Notch signaling is known to regulate both active and quiescent states of somatic stem cells, but how it regulates these different states is unknown. Recent studies revealed that the Notch effector Hes1 is expressed differently during the active and quiescent states during neurogenesis and myogenesis: high in the quiescent state and oscillatory in the active state. When the Hes1 expression level is high, both Ascl1 and MyoD expression are continuously suppressed. By contrast, when Hes1 expression oscillates, it periodically represses expression of the neurogenic factor Ascl1 and the myogenic factor MyoD, thereby driving Ascl1 and MyoD oscillations. High levels of Hes1 and the resultant Ascl1 suppression promote the quiescent state of neural stem cells, while Hes1 oscillation-dependent Ascl1 oscillations regulate their active state. Similarly, in satellite cells of muscles, known adult muscle stem cells, high levels of Hes1 and the resultant MyoD suppression seem to promote their quiescent state, while Hes1 oscillation-dependent MyoD oscillations activate their proliferation and differentiation. Therefore, the expression dynamics of Hes1 is a key regulatory mechanism of generating and maintaining active/quiescent stem cell states.     

Alpinate Oxyphyllae extracts enhance the longevity and homing of mesenchymal stem cells and augment their protection against senescence in H9c2 cells

Adipose-derived mesenchymal stem cells (ADMSCs) are easily accessible and are attractive mesenchymal stem cells for use in regenerative medicine; however their application is frequently restricted due to various challenges present in the environment they are administered. Therefore ADMSCs are preferably preconditioned with various stimulating factors to overcome the barriers developed in any pathological conditions. Here we used ADMSCs from rat adipose based on the abundance of positive markers and preconditioned the cells with extracts from Alpinate Oxyphyllae Fructus (AOF), a traditional Chinese herb used for antiaging, associated various health benefits. The preconditioned stem cells were tested for their potential to drive H9c2 from doxorubicin (Dox)-induced aging. The AOF-treated stem cells enriched stemness in ADMSCs with respect to their stem cells' positive marker, and enhanced their longevity mechanism and elevated the stem cell homing-associated C-X-C chemokine receptor type 7 (CXCR7). The AOF preconditioned stem cells, when cocultured with H9c2 cells, showed effective protection to Dox-induced senescence and stem cell homing to damaged H9c2 cells. The presence of AOF provided greater protective effects in the Dox environment. In addition, AOF-pretreated ADMSCs showed enhanced migration than those treated with AOF in Dox environment. Therefore, our results show that administration of AOF preconditioned stem cells is potentially an effective strategy in the management of aging-associated cardiac disorders.     

Influence of Regulatory Genes of Type 1 Human Immunodeficiency Virus on Proliferation and Differentiation of Murine Embryonic Stem Cells

Spontaneous formation of embryoid bodies and subsequent differentiation of some cells into cardiomyocytes were demonstrated on murine embryonic stem cells of R1 line. The lines of embryonic stem cells were obtained that had been transfected with genetic constructs carrying expressing regulatory genes of the human immunodeficiency virus tat and nef and green protein gene (GFP). The transfection of embryonic stem cells with the gene tat stimulated their proliferative activity, while this activity decreased in the cells transfected with the gene nef. The time necessary for the formation of embryoid bodies by all lines of transfected cells was similar to that in the control cells. In the cultures of cells transfected with nef and tat, the number of embryoid bodies and the percentage of embryoid bodies with contracting cardiomyocytes were higher and lower than in the control, respectively. Thus, an inverse correlation was observed between the effects of regulatory genes of the human immunodeficiency virus on proliferation and differentiation embryonic stem cells.     

Expression patterns of germ line specific genes in mouse and human pluripotent stem cells are associated with regulation of ground and primed state of pluripotency

One of the main criteria of pluripotency is ability of cell lines to differentiate into the germ line. Pluripotent stem cell lines in ground state of pluripotency differ from the lines in primed state by their ability to give rise to the mature gametes. To understand molecular mechanisms involved in regulation of different states of pluripotency we investigated the expression patterns of germ line specific genes in different type pluripotent stem cells and mouse and human embryonic teratocarcinoma cells. We found that pluripotent stem cells in vitro, in blastocyst and gonocytes at stage E13.5 had similar expression patterns in contrast to the epiblast cells at stage E6.5. Quantitative real time PCR analysis showed that Vasa/Ddx4 expression in mouse and human embryonic stem cells was significantly lower than in blastocyst and gonocytes. Moreover, Vasa/Ddx4 and E-ras expression was significantly higher in mouse embryonic stem cells than in human embryonic stem cells. Our analysis of germ line specific gene expression in differentiating mouse embryonic stem and embryonic germ cells as well as in mouse embryonic teratocarcinoma cells maintained under conditions promoting cell reprogramming from primed to ground state of pluripotency (2i + LIF) revealed that only pluripotent stem cells are able to regulate the expression level of Oct4 and Vasa/Ddx4 and restore initial ground state, while in embryonic teratocarcinoma cells the expression level of these genes remained unchanged. We suggest that expression patterns of germ lines specific genes, in particular of Vasa/Ddx4, can underlie the regulation of ground and primed states of pluripotency.