Functional Properties Of Stem Cells Research Articles

Age-related reduction in the functional properties of adult stem cells located in the peripheral region of human retinal pigment epithelium.

Adult stem cells (SCs) with self-renewal and multilineage potential have been reported upon culturing human retinal pigment epithelial (RPE) cells. The current study aimed to identify the location of SCs in human RPE and to elucidate the age-related changes. Peripheral, equatorial, and central RPE cells from donors of three age groups were analyzed for their sphere-forming, clonal, and label-retaining cell properties. Furthermore, native human RPE flatmounts were immunostained for SC and proliferating cell markers. Cells with higher sphere-forming and clonal ability were identified only in young donors (<30 years) and were restricted to the periphery. Upon culturing, cells from peripheral and equatorial regions had the label-retaining cell (LRC) property. With aging, the LRCs were restricted to the periphery and were reduced. In young donors, Ki67+ proliferating cells were not observed in native RPE. However, such cells were observed in the peripheral RPE of older donors correlating with the need for regeneration. The native RPE cells were negative for SC marker expression. The above findings highlighted the presence of SCs with the ability to proliferate in the peripheral RPE and a reduction in these functional properties of SCs with aging.

Self-Assembled Copolymeric Nanowires as a New Class of 3D Scaffold for Stem Cells Growth and Proliferation

Stem cell therapy has emerged as the most vibrant area of research, due to the capacity of stem cells for self-renewal and differentiation into different types of cell lines upon their culture. But lately, scientists become increasingly aware of the limitations of conventional 2D culture and stem cell culture media, due to several key drawbacks associated with this model, such as immune response upon transplantation, animal pathogen contamination, and complication, during developmental studies due to undefined factors in the cultural media. In this study, an attempt has been made to develop a new type of polymeric 3D scaffold based on the self-assembly of a star-like amphiphilic copolymer of poly(caprolactone)–poly(ethylene oxide) unit into nanowires (nanofibers), that have a scale similar to the native extracellular matrix and are capable of mimicking the extracellular microenvironment where the functional properties of stem cells can be observed and manipulated. The obtained data showed that polymeric-based nanofibers can be used as a 3D scaffold for mouse embryonic stem cells (mESCs) growth without losing their stem cell phenotype. The results obtained suggest that the polymeric 3D scaffolds (nanofibers) not only support stem cells’ growth and proliferation but also preserve the mESC pluripotency.

THE CLINICAL SIGNIFICANCE OF STEM CELLS IN CERVICAL DISEASES

Stem cells are cells capable of self-sustaining asymmetric division, resulting in one daughter cell and one progenitor cell from one stem cell. The features of stem cells are the ability to self-renew and differentiate into various cells, ensure hemostasis of tissues, as well as proliferative reserve. The maintenance and functional properties of stem cells are regulated by signals from the stem cell niche, i.e. from their local microenvironment, which contributes to adequate self-renewal and differentiation. Therefore, niches provide regulation of the number of stem cells in the body and protect the body from their excessive proliferation. Structurally, the stem niche is a combination of all factors that ensure the viability and self-reproduction of stem cells and the differentiation of daughter progenitor cells. An example of a stem cell niche can be the transition zone (TZs) - the junction of different types of epithelial tissue. It is believed that carcinogenesis can be associated with these zones: the transition region of the glandular and squamous epithelium in the conjunctiva, the cervical glands of the stomach, in the esophagus, in the lungs (bronchioalveolar transition zone), in the cervix (endo-ectocervical transition). This article discusses modern approaches to the search for significant markers of the state of stem cell niche and tumor stem cells of cervical cancer, which will be of great importance in clinical practice for the timely detection of oncological processes, even before their phenotypic manifestation.

A standard procedure for lentiviral-mediated labeling of murine mesenchymal stromal cells in vitro.

Tracking of stem cells after transplantation is effectively performed in vivo with imaging systems, assuming the cells are adequately labeled to facilitate their recognition. This study aimed to optimize a protocol for fluorescent labeling of mesenchymal stromal cells (MSCs) in vitro, by using a third-generation lentiviral system. Basically, 293T cells are seeded in high-glucose Dulbecco's modified Eagle medium with 10% FBS one day before transfection. Transfection is done for 24h using a mix of transfer, packaging, regulatory, and envelope plasmids, in molar ratio of 4:2:1:1, respectively. After transfection, the cells are further cultured for two days. During this period, the viral medium is harvested two times, at 24-h intervals, with the first round being stored at 4°C until the second round is completed. The pooled viral medium is frozen in single-use aliquots. MSCs are transduced with 25 multiplicity of infection (MOI) and one day later the cells are passaged at standard seeding density and further grown for three days, when the fluorescence reach the maximum level. Our protocol provides particular experimental details for permanent MSC labeling that makes the procedure highly effective for therapeutic purposes, without affecting the functional properties of stem cells.

Stem cell-associated heterogeneity in Glioblastoma results from intrinsic tumor plasticity shaped by the microenvironment

The identity and unique capacity of cancer stem cells (CSC) to drive tumor growth and resistance have been challenged in brain tumors. Here we report that cells expressing CSC-associated cell membrane markers in Glioblastoma (GBM) do not represent a clonal entity defined by distinct functional properties and transcriptomic profiles, but rather a plastic state that most cancer cells can adopt. We show that phenotypic heterogeneity arises from non-hierarchical, reversible state transitions, instructed by the microenvironment and is predictable by mathematical modeling. Although functional stem cell properties were similar in vitro, accelerated reconstitution of heterogeneity provides a growth advantage in vivo, suggesting that tumorigenic potential is linked to intrinsic plasticity rather than CSC multipotency. The capacity of any given cancer cell to reconstitute tumor heterogeneity cautions against therapies targeting CSC-associated membrane epitopes. Instead inherent cancer cell plasticity emerges as a novel relevant target for treatment.

Hypoxia-Activated PI3K/Akt Inhibits Oxidative Stress via the Regulation of Reactive Oxygen Species in Human Dental Pulp Cells.

In order to use stem cells as a resource for tissue regeneration, it is necessary to induce expansion in vitro. However, during culture, stem cells often lose functional properties and become senescent. Increasing evidence indicates that hypoxic preconditioning with physiological oxygen concentration can maintain the functional properties of stem cells in vitro. The purpose of the current study was to test the hypothesis that hypoxic preconditioning with physiological oxygen concentration can maintain the functional properties of stem cells in culture by reducing oxidative stress. In vitro studies were performed in primary human dental pulp cells (hDPCs). Reduced levels of oxidative stress and increased cellular “stemness” in response to physiological hypoxia were dependent upon the expression of reactive oxygen species (ROS). Subsequently, RNA-sequencing analysis revealed the increased expression of phosphoinositide 3-kinase (PI3K)/Akt signaling in culture, a pathway which regulates oxidative stress. Furthermore, we found evidence that PI3K/Akt signaling might affect intracellular ROS production by negatively regulating expression of the downstream protein Forkhead Box Protein O1 (FOXO1) and Caspase 3. Collectively, our data show that the PI3K/Akt pathway is activated in response to hypoxia and inhibits oxidative stress in a ROS-dependent manner. This study identified redox-mediated hypoxic preconditioning regulatory mechanisms that may be significant for tissue regeneration.

Off-label use of adipose-derived stem cells.

BackgroundAdipose-derived stem cells (ASCs) have a broad range of clinical applications. The ease of cell harvest and high yield with minimal donor-site morbidity makes adipose tissue an ideal source of stem cells. Further, the multi-lineage potential of these cells present significant opportunities within the field of tissue engineering, with studies successfully demonstrating their ability to produce a range of tissue types.Materials and methodsLiterature review of publications on the use of ASCs, in the context of current European and US regulations.ResultsAccording to European and US regulations, many clinical trials reported in literature to date could be considered off-label.ConclusionIn Europe, clinical trials involving cultured ASCs and/or the use of collagenase, which causes changes in the structural and functional properties of stem cells, and/or ASCs application in non-homologous tissue, should be considered off-label. ASCs should be non-cultured, isolated mechanically, and used only in the subcutaneous tissue.

Stem cell hunt in NHL.

Non-Hodgkin Lymphoma (NHL) is a heterogenous disease consisting of at least 40 different entities that are defined mainly by their clinical and histopathological presentation, their underlying genetic abnormalities but also their presumed cell of origin [1]. The latter can be of B- or T-cell type and derived from different stages of the differentiation process. B-cells are predisposed to DNA alterations including somatic hypermutation, class switch recombination and antigen receptor rearrangement. This innate genetic instability may be one of the reasons why B-cell NHL is the most frequently diagnosed lymphoma in the Western World; many B-cell NHLs arise from germinal center (GC) cells or cells that have just left the GC [2]. Hence, most NHL (including T-cell NHL) are considered to arise from mature, peripheral lymphoid cells. In contrast, both T- and B-lymphoblastic lymphoma/leukaemia cells arise from immature precursors that are found in the thymus and in the bone marrow, respectively. Based on these findings one may hypothesize that the differentiation status of the tumor cell defines their cell of origin in healthy tissue. However, it is also possible that mature lymphoid cells change differentiation status when they become neoplastic and/or that differentiation processes contribute to disease pathogenesis with the seeds of malignancy planted much earlier in their lifespan. We have shown in a specific entity of T-NHL that some of the tumor cells have a mature cell surface phenotype despite having the ‘ghostly’ genetic appearance of a primitive origin, supporting the idea of cell plasticity in the context of neoplasia [3]. Anaplastic Large Cell Lymphoma (ALCL) is a peripheral T-cell lymphoma; tumour cells are found in the periphery at nodal and extranodal sites, express proteins associated with a cytotoxic T-cell function and exhibit rearrangements of the T-cell receptor (TCR) at the molecular level (the TCR is rarely expressed on the surface of ALCL cells) [3]. However, contrary to popular belief, at least in paediatric ALCL, mediastinal involvement is not infrequent (50%) implicating the thymus, the organ of T-cell development in disease pathogenesis [4]. We show that a gene signature enriched in early thymic progenitors can be detected in a subset of ALCL tumour cells which functionally act as tumour propagating or cancer stem cells (CSC) [3]. The ALCL CSC were isolated using the Side Population (SP) technique which relies on the functional properties of stem cells, i.e. quiescence and expression of efflux pumps presumably evolutionarily conserved to protect the stem cell compartment. This technique has been applied to a number of cancers and was originally developed to enrich for haemopoietic stem cells in murine bone marrow [5]. We show that the ALCL SP cells not only give rise to the bulk tumour population whilst self-replicating to a discrete level but also produce as yet unidentified soluble factors that support the growth of the tumour as a whole [3]. Therefore we hypothesize that a thymic origin may apply to this disease and that these ‘primed’ T-cells egress the thymus and are able to survive and circulate in the periphery to eventually transform as a result of yet to be identified events giving rise to ALCL CSC. Whether distinct clones of ALCL CSC also exist remains to be determined and whilst to date we have been able to identify these cells by means of their functional attributes, in depth mechanistic analyses still need to be performed. It is clear now that at least a limited hierarchy exists and given the postulated thymic origin of these cells it is very likely that tumour propagating cells may reside in the thymus to seed disease relapse, an event that is common in children with ALCL [6]. Figure 1 ALCL cancer stem cells (CSC) have a potential thymic origin

Prolonged Antiretroviral Therapy Preserves HIV-1-Specific CD8 T Cells with Stem Cell-Like Properties.

HIV-1-specific CD8 T cells can influence HIV-1 disease progression during untreated HIV-1 infection, but the functional and phenotypic properties of HIV-1-specific CD8 T cells in individuals treated with suppressive antiretroviral therapy remain less well understood. Here we show that a subgroup of HIV-1-specific CD8 T cells with stem cell-like properties, termed T memory stem cells (TSCM cells), is enriched in patients receiving suppressive antiretroviral therapy compared with their levels in untreated progressors or controllers. In addition, a prolonged duration of antiretroviral therapy was associated with a progressive increase in the relative proportions of these stem cell-like HIV-1-specific CD8 T cells. Interestingly, the proportions of HIV-1-specific CD8 TSCM cells and total HIV-1-specific CD8 TSCM cells were associated with the CD4 T cell counts during treatment with antiretroviral therapy but not with CD4 T cell counts, viral loads, or immune activation parameters in untreated patients, including controllers. HIV-1-specific CD8 TSCM cells had increased abilities to secrete interleukin-2 in response to viral antigen, while secretion of gamma interferon (IFN-γ) was more limited in comparison to alternative HIV-1-specific CD8 T cell subsets; however, only proportions of IFN-γ-secreting HIV-1-specific CD8 TSCM cells were associated with CD4 T cell counts during antiretroviral therapy. Together, these data suggest that HIV-1-specific CD8 TSCM cells represent a long-lasting component of the cellular immune response to HIV-1 that persists in an antigen-independent fashion during antiretroviral therapy but seems unable to survive and expand under conditions of ongoing viral replication during untreated infection. Memory CD8 T cells that imitate the functional properties of stem cells to maintain lifelong cellular immunity have been hypothesized for many years, but only recently have such cells, termed T memory stem cells (TSCM cells), been physically identified and isolated in humans, mice, and nonhuman primates. Here, we investigated whether cellular immune responses against HIV-1 include such T memory stem cells. Our data show that HIV-1-specific CD8 T memory stem cells are detectable during all stages of HIV-1 infection but occur most visibly at times of prolonged viral antigen suppression by antiretroviral combination therapy. These cells may therefore be particularly relevant for designing antiviral immune defense strategies against the residual reservoir of HIV-1-infected cells that persists despite treatment and leads to viral rebound upon treatment discontinuation.

Aldehyde dehydrogenase isozymes: markers of cancer stem cells in human melanoma.

Aldehyde dehydrogenase isozymes: markers of cancer stem cells in human melanoma.

Epithelial-Mesenchymal Transition and the Stem Cell Phenotype

Epithelial-mesenchymal transition (EMT) is a developmental process in which epithelial cells acquire the motile, migratory properties of mesenchymal cells. In a recent issue of Cell, Mani et al. (2008) show that induction of EMT stimulates cultured breast cells to adopt characteristics of stem cells.

Neural stem cells: an overview.

This review focuses on the nature and functional properties of stem cells of the adult mammalian central nervous system (CNS). It has recently been shown that cell turnover, including neurons, does occur in the mature CNS, thanks to the persistence of precursor cells that possess the functional characteristics of bona-fide neural stem cells (NSCs) within restricted brain areas. We discuss how the subventricular zone of the forebrain (SVZ) is the most active neurogenetic area and the richest source of NSCs. These NSCs ensure a life-long contribution of new neurons to the olfactory bulb and, when placed in culture, can be grown and extensively expanded for months, allowing the generation of stem cell lines, which maintain stable and constant functional properties. A survey of the differentiation potential of these NSCs, both in vitro and in vivo, outlines their extreme plasticity that seems to outstretch the brain boundaries, so that these neuroectodermal stem cells may give rise to cells that derive from developmentally distinct tissues. A critical discussion of the latest, controversial findings regarding this surprising phenomenon is provided.

Sox2 regulatory sequences direct expression of a (beta)-geo transgene to telencephalic neural stem cells and precursors of the mouse embryo, revealing regionalization of gene expression in CNS stem cells.

Sox2 is one of the earliest known transcription factors expressed in the developing neural tube. Although it is expressed throughout the early neuroepithelium, we show that its later expression must depend on the activity of more than one regionally restricted enhancer element. Thus, by using transgenic assays and by homologous recombination-mediated deletion, we identify a region upstream of Sox2 (-5.7 to -3.3 kb) which can not only drive expression of a (beta)-geo transgene to the developing dorsal telencephalon, but which is required to do so in the context of the endogenous gene. The critical enhancer can be further delimited to an 800 bp fragment of DNA surrounding a nuclease hypersensitive site within this region, as this is sufficient to confer telencephalic expression to a 3.3 kb fragment including the Sox2 promoter, which is otherwise inactive in the CNS. Expression of the 5.7 kb Sox2(beta)-geo transgene localizes to the neural plate and later to the telencephalic ventricular zone. We show, by in vitro clonogenic assays, that transgene-expressing (and thus G418-resistant) ventricular zone cells include cells displaying functional properties of stem cells, i.e. self-renewal and multipotentiality. We further show that the majority of telencephalic stem cells express the transgene, and this expression is largely maintained over two months in culture (more than 40 cell divisions) in the absence of G418 selective pressure. In contrast, stem cells grown in parallel from the spinal cord never express the transgene, and die in G418. Expression of endogenous telencephalic genes was similarly observed in long-term cultures derived from the dorsal telencephalon, but not in spinal cord-derived cultures. Thus, neural stem cells of the midgestation embryo are endowed with region-specific gene expression (at least with respect to some networks of transcription factors, such as that driving telencephalic expression of the Sox2 transgene), which can be inherited through multiple divisions outside the embryonic environment.

Stem cells: the intestinal stem cell as a paradigm.

Stem cell research provides a foundation for therapeutic advancement in oncology, clinical genetics and a diverse array of degenerative disorders. For example, the elucidation of pathways governing proliferative regulation and differentiation within cellular systems will result in medical strategies aimed at the root cause of cancer. At present the characterization of reliable stem cell markers is the immediate aim in this particular field. Over the past 30 years investigators have determined many of the physical and functional properties of stem cells through careful and imaginative experimentation. Intestinal stem cells reside at the crypt base and give rise to all cell types found within the crypt. They readily undergo altruistic apoptosis in response to toxic stimuli although their progeny are hardier and will regain stem cell function to repopulate the tissue compartment, giving rise to the concept of a proliferative hierarchy. Contention exists when deciding whether the full complement of cells within a crypt is derived from either a single or multiple stems. Evidence has also arisen to challenge the long held view that colorectal tumours arise from a single mutated stem cell, as early adenomas from a human XO/XY mosaic contained distinct clones. Mechanisms governing the stem cell cycle and subsequent proliferative activity largely remain obscure. The adenomatous polyposis coli gene product has, however, been shown to promote the degradation of beta-catenin, an enhancer of cell proliferation, thereby downregulating this activity in healthy individuals.

The intestinal epithelial stem cell: the mucosal governor.

All epithelial cells in the small and large intestine are thought to originate from stem cells located towards the base of the crypts of Lieberkühn. To-date, there are no specific intestinal stem cell markers, hence stem cell properties can only be inferred. A range of experimental techniques have been employed including cell position mapping, radiation regeneration (clonogenic) assays, chimeric and transgenic mice. This review discusses the implications of experiments performed using these techniques in order to deduce the number, location and functional properties of stem cells. Stem cell homeostasis is maintained by cell proliferation and death 'through apoptosis'. The various growth and matrix factors and genes which may control these processes, and be important for stem cell function, are discussed along with their carcinogenic and clinical implications.