Control Stem Cell Behavior Research Articles

Association of Gankyrin and Stemness Factor Expression in Human Colorectal Cancer

It is widely accepted that the adenoma-carcinoma sequence represents the process by which most colorectal cancers (CRCs) arise. Although gankyrin is overexpressed in CRC tissues, its roles in the initiation step of colorectal carcinogenesis remain largely unexplored. We investigated the expression of gankyrin and stemness factors in human colorectal adenomas, precancerous lesions, as well as CRC tissues to assess its involvement in colorectal carcinogenesis. Expression of several molecules including gankyrin and certain stemness factors was compared in 50 pairs of adenoma and surrounding normal mucosa using real-time quantitative polymerase chain reaction and in 30 CRC tissues using immunohistochemistry. In CRC specimens, expression of CD133, a cancer stem cell marker, was significantly correlated with gankyrin expression. Gankyrin knockdown decreased the expression of vascular endothelial growth factor (VEGF) and stemness factors such as Nanog and Oct-4 in colorectal cancer cells. Expression of gankyrin and these stemness factors was significantly higher in adenomas than in the surrounding normal mucosa. Importantly, a significant correlation was observed between the expression of gankyrin, VEGF, and Nanog in colorectal adenomas. In CRC development, gankyrin would control stem cell behavior by regulating the expression of stemness factors.

2D and 3D Nanopatterning of Titanium for Enhancing Osteoinduction of Stem Cells at Implant Surfaces

The potential for the use of well-defined nanopatterns to control stem cell behaviour on surfaces has been well documented on polymeric substrates. In terms of translation to orthopaedic applications, there is a need to develop nanopatterning techniques for clinically relevant surfaces, such as the load-bearing material titanium (Ti). In this work, a novel nanopatterning method for Ti surfaces is demonstrated, using anodisation in combination with PS-b-P4VP block copolymer templates. The block copolymer templates allows for fabrication of titania nanodot patterns with precisely controlled dimensions and positioning which means that this technique can be used as a lithography-like patterning method of bulk Ti surfaces on both flat 2D and complex shaped 3D surfaces. In vitro studies demonstrate that precise tuning of the height of titania nanodot patterns can modulate the osteogenic differentiation of mesenchymal stem cells. Cells on both the 8 nm and 15 nm patterned surfaces showed a trend towards a greater number of the large, super-mature osteogenic focal adhesions than on the control polished Ti surface, but the osteogenic effect was more pronounced on the 15 nm substrate. Cells on this surface had the longest adhesions of all and produced larger osteocalcin deposits. The results suggest that nanopatterning of Ti using the technique of anodisation through a block copolymer template could provide a novel way to enhance osteoinductivity on Ti surfaces.

Live imaging reveals active infiltration of mitotic zone by its stem cell niche.

Stem cells niches are increasingly recognized as dynamic environments that play a key role in transducing signals that allow an organism to exert control on its stem cells. Live imaging of stem cell niches in their in vivo setting is thus of high interest to dissect stem cell controls. Here we report a new microfluidic design that is highly amenable to dissemination in biology laboratories that have no microfluidics expertise. This design has allowed us to perform the first time lapse imaging of the C. elegans germline stem cell niche. Our results show that this niche is strikingly dynamic, and that morphological changes that take place during development are the result of a highly active process. These results lay the foundation for future studies to dissect molecular mechanisms by which stem cell niche morphology is modulated, and by which niche morphology controls stem cell behavior.

Evolution of human longevity: lessons from Hydra

Comment on: Boehm AM et al. FoxO is a critical regulator of stem cell maintenance in immortal Hydra. Proc Natl Acad Sci USA. 2012; 109:19697-19702.

Designing Biomaterials To Direct Stem Cell Fate

As stem cells are a cornerstone of regenerative medicine, research efforts have been extensively focused on controlling their self-renewal and differentiation. It is well-known that stem cells are tightly regulated by a combination of physical and chemical factors from their complex extracellular surroundings; thus, conventional cell culture approaches based purely on using soluble factors to direct stem cell fate have resulted in limited success. To account for the complexities of native stem-cell niches, biomaterials are actively investigated as artificial extracellular matrices in order to mimic the natural microenvironment. This Perspective highlights important areas related to the design of biomaterials to control stem cell behavior, such as cell-responsive ligands, mechanical signals, and delivery of soluble factors.

Sex-lethal enables germline stem cell differentiation by down-regulating Nanos protein levels during Drosophila oogenesis

Drosophila ovarian germ cells require Sex-lethal (Sxl) to exit from the stem cell state and to enter the differentiation pathway. Sxl encodes a female-specific RNA binding protein and in somatic cells serves as the developmental switch gene for somatic sex determination and X-chromosome dosage compensation. None of the known Sxl target genes are required for germline differentiation, leaving open the question of how Sxl promotes the transition from stem cell to committed daughter cell. We address the mechanism by which Sxl regulates this transition through the identification of nanos as one of its target genes. Previous studies have shown that Nanos protein is necessary for GSC self-renewal and is rapidly down-regulated in the daughter cells fated to differentiate in the adult ovary. We find that this dynamic expression pattern is limited to female germ cells and is under Sxl control. In the absence of Sxl, or in male germ cells, Nanos protein is continuously expressed. Furthermore, this female-specific expression pattern is dependent on the presence of canonical Sxl binding sites located in the nanos 3' untranslated region. These results, combined with the observation that nanos RNA associates with the Sxl protein in ovarian extracts and loss and gain of function studies, suggest that Sxl enables the switch from germline stem cell to committed daughter cell by posttranscriptional down-regulation of nanos expression. These findings connect sexual identity to the stem cell self-renewal/differentiation decision and highlight the importance of posttranscriptional gene regulatory networks in controlling stem cell behavior.

The Drosophila BCL6 homolog ken and barbie promotes somatic stem cell self-renewal in the testis niche

Stem cells sustain tissue regeneration by their remarkable ability to replenish the stem cell pool and to generate differentiating progeny. Signals from local microenvironments, or niches, control stem cell behavior. In the Drosophila testis, a group of somatic support cells called the hub creates a stem cell niche by locally activating the Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) pathway in two adjacent types of stem cells: germline stem cells (GSCs) and somatic cyst stem cells (CySCs). Here, we find that ken and barbie (ken) is autonomously required for the self-renewal of CySCs but not GSCs. Furthermore, Ken misexpression in the CySC lineage induces the cell-autonomous self-renewal of somatic cells as well as the nonautonomous self-renewal of germ cells outside the niche. Thus, Ken, like Stat92E and its targets ZFH1 (Leatherman and Dinardo, 2008) and Chinmo (Flaherty et al., 2010), is necessary and sufficient for CySC renewal. However, ken is not a JAK-STAT target in the testis, but instead acts in parallel to Stat92E to ensure CySC self-renewal. Ken represses a subset of Stat92E targets in the embryo (Arbouzova et al., 2006) suggesting that Ken maintains CySCs by repressing differentiation factors. In support of this hypothesis, we find that the global JAK-STAT inhibitor Protein tyrosine phosphatase 61F (Ptp61F) is a JAK-STAT target in the testis that is repressed by Ken. Together, our work demonstrates that Ken has an important role in the inhibition of CySC differentiation. Studies of ken may inform our understanding of its vertebrate orthologue B-Cell Lymphoma 6 (BCL6) and how misregulation of this oncogene leads to human lymphomas.

Abstract 4187: A novel genome-wide assay identifies chromatin domains involved in stem cell behavior

Abstract The activity of DNA regulatory elements are often controlled by chromatin structure. Active regulatory elements are located within “open” or “accessible” chromatin. If a regulatory element is in a “closed” or “inaccessible” chromatin environment it is functionally silenced. We find that when permeabilized cells are treated with nuclease in situ, small DNA fragments, corresponding to accessible chromatin, can be readily isolated. These small DNA fragments, when analyzed by next-generation sequencing, provide a global map of active genomic regulatory domains. We used this technology to identify chromatin domains involved in stem cell behavior using NTera2 cells as a model system. By comparing differentiated and undifferentiated NTera2 cells we identified more than 500 genomic regions that are accessible only in undifferentiated cells. We then used these results, together with ENCODE data analyzing human embryonic stem cells and human cancer cells, to identify genomic regulatory regions that are active only in stem cells. Importantly, many of these regions are proximal to genes that are associated with cell “stemness” (NANOG and LIN28 are examples). In addition, several uncharacterized genes that have RNA expression characteristics similar to NANOG and LIN28 were identified. Interestingly, several stem-cell specific active regulatory domains were found adjacent to previously uncharacterized, stem-cell specific non-coding RNA transcript regions. These findings demonstrate that this approach can serve as a new discovery tool to identify chromatin regions that control stem cell behavior. In addition, chromatin regions associated with other biological or pathological states could also be identified using this approach. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4187. doi:1538-7445.AM2012-4187

Using polymeric materials to control stem cell behavior for tissue regeneration.

Patients with organ failure often suffer from increased morbidity and decreased quality of life. Current strategies of treating organ failure have limitations, including shortage of donor organs, low efficiency of grafts, and immunological problems. Tissue engineering emerged about two decades ago as a strategy to restore organ function with a living, functional engineered substitute. However, the ability to engineer a functional organ is limited by a limited understanding of the interactions between materials and cells that are required to yield functional tissue equivalents. Polymeric materials are one of the most promising classes of materials for use in tissue engineering, due to their biodegradability, flexibility in processing and property design, and the potential to use polymer properties to control cell function. Stem cells offer potential in tissue engineering because of their unique capacity to self-renew and differentiate into neurogenic, osteogenic, chondrogenic, and myogenic lineages under appropriate stimuli from extracellular components. This review examines recent advances in stem cell-polymer interactions for tissue regeneration, specifically highlighting control of polymer properties to direct adhesion, proliferation, and differentiation of stem cells, and how biomaterials can be designed to provide some of the stimuli to cells that the natural extracellular matrix does.

Epithelial stem cells, wound healing and cancer

It is well established that tissue repair depends on stem cells and that chronic wounds predispose to tumour formation. However, the association between stem cells, wound healing and cancer is poorly understood. Lineage tracing has now shown how stem cells are mobilized to repair skin wounds and how they contribute to skin tumour development. The signalling pathways, including WNT and Hedgehog, that control stem cell behaviour during wound healing are also implicated in tumour formation. Furthermore, tumorigenesis and wound repair both depend on communication between epithelial cells, mesenchymal cells and bone marrow-derived cells. These studies suggest ways to harness stem cells for wound repair while minimizing cancer risk.

Changes in embryonic stem cell colony morphology and early differentiation markers driven by colloidal crystal topographical cues

The use of materials properties to guide cell behaviour is an attractive option for regenerative medicine, where controlling stem cell behaviour is important for the establishment of a functioning cell population. A wide range of materials properties have been shown to influence many types of cells but little is known about the effects of topography on embryonic stem cells (ESCs). In order to advance this knowledge, we synthesised and characterised substrates formed of silica colloidal crystal (SCC) microspheres to present highly ordered and reproducible topographical features from 120-600 nm in diameter. We found that, compared to cells cultured on flat glass, cells cultured on the SCC substrates retained transcription of stem cell (Dppa5a, Nanog, and Pou5f1) and endoderm (Afp, Gata4, Sox17, and Foxa2) markers more similar to undifferentiated ESCs, suggesting the substrates are restricting differentiation, particularly towards the endoderm lineage. Additionally, five days after seeding, we observed strikingly different colony morphology, with cells on the SCC substrates growing in spherical colonies approximately ten cells thick, while cells on glass were growing in flat monolayers. Colonies on the SCC substrates developed a central pit, which was never observed in cells cultured on glass, and expressed proteins related to epithelialisation. Together, these data demonstrate the potential of using topographical cues to control stem cell behaviour in vitro.

Identification and Characterization of Human Leukemia Stem Cell Functional Regulators

Abstract 2955Emerging evidence shows that many cancers are organized as cellular hierarchies sustained by a subpopulation of cancer stem cells (CSC). It is thought that CSC properties influence therapy response, disease relapse, and overall survival. However, little is currently known about the molecular pathways that control stem cell behaviour. Our goal is to identify and characterize the molecular regulatory networks that govern the self-renewal and developmental program of human leukemia stem cells (LSC).To expand our understanding of LSC function, we selected genes from our previously established LSC transcriptional program as novel candidate regulators of stem cell function. The LSC transcriptional program was identified by fractionation of 16 primary human acute myeloid leukemia (AML) samples into four populations that were subjected to sensitive in vivo LSC assays. mRNA expression analysis was performed on each fraction and a global LSC-specific signature was determined from functionally defined LSC fractions. Using similar methodology, a hematopoietic stem cell (HSC) enriched gene signature was established from human cord blood. Bioinformatic analysis identified a core transcriptional program that LSCs and HSCs share, revealing the molecular machinery that underlies stemness properties. The LSC and HSC signatures have prognostic significance independent of other factors when validated on a large cohort of cytogenetically normal AML patients. We have begun experiments to determine the role of 17 candidate stem cell regulators using functional in vivo and in vitro assays.We first examined the effect of candidate gene overexpression in normal HSC using xenograft assays. Lineage negative cord blood cells were transduced with lentiviral overexpression vectors, injected into immune-deficient mice and engraftment of human cells was measured after 12 weeks. In the preliminary round of in vivo analysis, over-expression of GPR56 increased the engraftment capability of HSCs in immune-deficient recipients compared to control (p=0.0019). GPR56 is involved in cell adhesion and differentiation and evidence is accumulating for a role in various cancers such as melanoma, esophageal cancer, and glioma. By qPCR analysis we observed that GPR56 mRNA is more highly expressed in both LSC-enriched and primitive normal hematopoietic cell populations, including highly purified HSC, compared to more mature cell populations such as AML blasts and normal lineage committed progenitor cells.In conclusion, our LSC and HSC data established that determinants of stemness influence clinical outcome of AML patients. We have identified multiple novel candidate stem cell-related genes and provided evidence for a role for GPR56 in hematopoietic stem cell regulation. Disclosures:No relevant conflicts of interest to declare.

ChemInform Abstract: Microenvironmental Regulation of Stem Cells by Biophysical and Material Cues

AbstractReview: 156 refs.

Abstract SY31-01: Combinatorial development of biomaterials for tissue engineering and drug delivery

Abstract High throughput, combinatorial approaches have revolutionized small molecule drug discovery. Here we describe our work on high throughput methods for developing and characterizing biomaterials for drug delivery and tissue engineering. We have developed automated methods allowing for rapid nanoliter scale synthesis of 1000's of biomaterials, as well as the testing of their chemical, material, and biological properties. These methods have also been applied towards the development of new methods to control stem cell behavior, as well as vehicles for drug delivery. In particular, these combinatorial libraries of different biomaterials have enabled new methods for microparticulate drug delivery, non-viral gene therapy, siRNA delivery, and vaccines. 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 SY31-01. doi:10.1158/1538-7445.AM2011-SY31-01

Mechanical stimuli differentially control stem cell behavior: morphology, proliferation, and differentiation

Mesenchymal stem cell (MSC) therapy has demonstrated applications in vascular regenerative medicine. Although blood vessels exist in a mechanically dynamic environment, there has been no rigorous, systematic analysis of mechanical stimulation on stem cell differentiation. We hypothesize that mechanical stimuli, relevant to the vasculature, can differentiate MSCs toward smooth muscle (SMCs) and endothelial cells (ECs). This was tested using a unique experimental platform to differentially apply various mechanical stimuli in parallel. Three forces, cyclic stretch, cyclic pressure, and laminar shear stress, were applied independently to mimic several vascular physiologic conditions. Experiments were conducted using subconfluent MSCs for 5days and demonstrated significant effects on morphology and proliferation depending upon the type, magnitude, frequency, and duration of applied stimulation. We have defined thresholds of cyclic stretch that potentiate SMC protein expression, but did not find EC protein expression under any condition tested. However, a second set of experiments performed at confluence and aimed to elicit the temporal gene expression response of a select magnitude of each stimulus revealed that EC gene expression can be increased with cyclic pressure and shear stress in a cell-contact-dependent manner. Further, these MSCs also appear to express genes from multiple lineages simultaneously which may warrant further investigation into post-transcriptional mechanisms for controlling protein expression. To our knowledge, this is the first systematic examination of the effects of mechanical stimulation on MSCs and has implications for the understanding of stem cell biology, as well as potential bioreactor designs for tissue engineering and cell therapy applications.

Presentation counts: microenvironmental regulation of stem cells by biophysical and material cues.

Stem cells reside in adult and embryonic tissues in a broad spectrum of developmental stages and lineages, and they are thus naturally exposed to diverse microenvironments or niches that modulate their hallmark behaviors of self-renewal and differentiation into one or more mature lineages. Within each such microenvironment, stem cells sense and process multiple biochemical and biophysical cues, which can exert redundant, competing, or orthogonal influences to collectively regulate cell fate and function. The proper presentation of these myriad regulatory signals is required for tissue development and homeostasis, and their improper appearance can potentially lead to disease. Whereas these complex regulatory cues can be challenging to dissect using traditional cell culture paradigms, recently developed engineered material systems offer advantages for investigating biochemical and biophysical cues, both static and dynamic, in a controlled, modular, and quantitative fashion. Advances in the development and use of such systems have helped elucidate novel regulatory mechanisms controlling stem cell behavior, particularly the importance of solid-phase mechanical and immobilized biochemical microenvironmental signals, with implications for basic stem cell biology, disease, and therapeutics.

Transmembrane potential of GlyCl-expressing instructor cells induces a neoplastic-like conversion of melanocytes via a serotonergic pathway.

SUMMARYUnderstanding the mechanisms that coordinate stem cell behavior within the host is a high priority for developmental biology, regenerative medicine and oncology. Endogenous ion currents and voltage gradients function alongside biochemical cues during pattern formation and tumor suppression, but it is not known whether bioelectrical signals are involved in the control of stem cell progeny in vivo. We studied Xenopus laevis neural crest, an embryonic stem cell population that gives rise to many cell types, including melanocytes, and contributes to the morphogenesis of the face, heart and other complex structures. To investigate how depolarization of transmembrane potential of cells in the neural crest’s environment influences its function in vivo, we manipulated the activity of the native glycine receptor chloride channel (GlyCl). Molecular-genetic depolarization of a sparse, widely distributed set of GlyCl-expressing cells non-cell-autonomously induces a neoplastic-like phenotype in melanocytes: they overproliferate, acquire an arborized cell shape and migrate inappropriately, colonizing numerous tissues in a metalloprotease-dependent fashion. A similar effect was observed in human melanocytes in culture. Depolarization of GlyCl-expressing cells induces these drastic changes in melanocyte behavior via a serotonin-transporter-dependent increase of extracellular serotonin (5-HT). These data reveal GlyCl as a molecular marker of a sparse and heretofore unknown cell population with the ability to specifically instruct neural crest derivatives, suggest transmembrane potential as a tractable signaling modality by which somatic cells can control stem cell behavior at considerable distance, identify a new biophysical aspect of the environment that confers a neoplastic-like phenotype upon stem cell progeny, reveal a pre-neural role for serotonin and its transporter, and suggest a novel strategy for manipulating stem cell behavior.

Neural stem cell adhesion and proliferation on phospholipid bilayers functionalized with RGD peptides

Peptide-functionalized materials show promise in controlling stem cell behavior by mimicking cell–matrix interactions. Supported lipid bilayers are an excellent platform for displaying peptides due to their ease of fabrication and low non-specific interactions with cells. In this paper, we report on the behavior of adult hippocampal neural stem cells (NSCs) on phospholipid bilayers functionalized with different RGD-containing peptides: either GGGNGEP RGDTYRAY (‘bsp-RGD(15)’) or G RGDSP. Fluid supported bilayers were prepared on glass surfaces by adsorption and fusion of small lipid vesicles incorporating synthetic peptide amphiphiles. NSCs adhered to bilayers with either GRGDSP or bsp-RGD(15) peptide. After 5 days in culture, NSCs formed neurosphere-like aggregates on GRGDSP bilayers, whereas on bsp-RGD(15) bilayers a large fraction of single adhered cells were observed, comparable to monolayer growth seen on laminin controls. NSCs retained their ability to differentiate into neurons and astrocytes on both peptide surfaces. This work illustrates the utility of supported bilayers in displaying peptide ligands and demonstrates that RGD peptides may be useful in synthetic culture systems for stem cells.

Nanog, Gli, and p53: a new network of stemness in development and cancer

Nanog and Hedgehog (HH) are both essential regulators of stemness by promoting self-renewal in embryonic stem cells, during early embryonal development (Mitsui et al, 2003) and in cancer cells (Jeter et al, 2009). Two groups have now shown that HH signalling regulates Nanog expression in neural stem cells in the adult brain and in brain tumours. In this issue of The EMBO Journal, Po et al (2010) and Zbinden et al (2010) demonstrate that downstream effectors in the HH pathway, Gli1 and Gli2 directly bind to the Nanog promoter and thus activate Nanog expression (Figure 1A). Po et al (2010) use the cerebellum as experimental paradigm (Figure 1C) and show that Nanog is highly expressed in stem cells derived from the postnatal cerebellum and in medulloblastoma, a malignant brain tumour arising from the cerebellum in children. This work is complemented by a study by Zbinden et al (2010), who use glial tumours and tumour spheres to demonstrate that the Gli-Nanog axis promotes stemness and growth in gliomas (Figure 1B).[GRAPHICS].

TSC1/2 tumour suppressor complex maintainsDrosophilagermline stem cells by preventing differentiation

Tuberous sclerosis complex human disease gene products TSC1 and TSC2 form a functional complex that negatively regulates target of rapamycin (TOR), an evolutionarily conserved kinase that plays a central role in cell growth and metabolism. Here, we describe a novel role of TSC1/2 in controlling stem cell maintenance. We show that in the Drosophila ovary, disruption of either the Tsc1 or Tsc2 gene in germline stem cells (GSCs) leads to precocious GSC differentiation and loss. The GSC loss can be rescued by treatment with TORC1 inhibitor rapamycin, or by eliminating S6K, a TORC1 downstream effecter, suggesting that precocious differentiation of Tsc1/2 mutant GSC is due to hyperactivation of TORC1. One well-studied mechanism for GSC maintenance is that BMP signals from the niche directly repress the expression of a differentiation-promoting gene bag of marbles (bam) in GSCs. In Tsc1/2 mutant GSCs, BMP signalling activity is downregulated, but bam expression is still repressed. Moreover, Tsc1 bam double mutant GSCs could differentiate into early cystocytes, suggesting that TSC1/2 controls GSC differentiation via both BMP-Bam-dependent and -independent pathways. Taken together, these results suggest that TSC prevents precocious GSC differentiation by inhibiting TORC1 activity and subsequently differentiation-promoting programs. As TSC1/2-TORC1 signalling is highly conserved from Drosophila to mammals, it could have a similar role in controlling stem cell behaviour in mammals, including humans.