Brain micro-ecologies: neural stem cell niches in the adult mammalian brain

Neurogenesis persists in two germinal regions in the adult mammalian brain, the subventricular zone of the lateral ventricles and the subgranular zone in the hippocampal formation. Within these two neurogenic niches, specialized astrocytes are neural stem cells, capable of self-renewing and generating neurons and glia. Cues within the niche, from cell-cell interactions to diffusible factors, are spatially and temporally coordinated to regulate proliferation and neurogenesis, ultimately affecting stem cell fate choices. Here, we review the components of adult neural stem cell niches and how they act to regulate neurogenesis in these regions.     

Enhancing stem cell survival in vivo for tissue repair

The ability to use progenitor cells for regenerative medicine remains an evolving but elusive clinical goal. A serious obstacle towards widespread use of stem cells for tissue regeneration is the challenges that face these cells when they are placed in vivo into a wound for therapy. These environments are hypoxic, acidic, and have an upregulation of inflammatory mediators creating a region that is hostile towards cellular survival. Within this environment, the majority of progenitor cells undergo apoptosis prior to participating in lineage differentiation and cellular integration. In order to maximize the clinical utility of stem cells, strategies must be employed to increase the cell's ability to survive in vivo through manipulation of both the stem cell and the surrounding environment. This review focuses on current advances and techniques being used to increase in vivo stem cell survival for the purpose of tissue regeneration.     

An emerging role for TOR signaling in mammalian tissue and stem cell physiology

The mammalian target of rapamycin (mTOR) is a kinase that responds to a myriad of signals, ranging from nutrient availability and energy status, to cellular stressors, oxygen sensors and growth factors. The finely tuned response of mTOR to these stimuli results in alterations to cell metabolism and cell growth. Recent studies of conditional knockouts of mTOR pathway components in mice have affirmed the role of mTOR signaling in energy balance, both at the cell and whole organism levels. Such studies have also highlighted a role for mTOR in stem cell homeostasis and lifespan determination. Here, we discuss the molecular mechanisms of TOR signaling and review recent in vitro and in vivo studies of mTOR tissue-specific activities in mammals.     

Advancing vascular tissue engineering: the role of stem cell technology

Atherosclerosis and heart disease are still the leading causes of morbidity and mortality worldwide. The lack of suitable autologous grafts has produced a need for artificial grafts but the patency of such grafts is limited compared to natural materials. Tissue engineering, whereby living tissue replacements can be constructed, has emerged as a solution to some of these difficulties. This, in turn, is limited by the availability of suitable cells from which to construct the vessels. The development of prosthesis using progenitor cells and switching these into endothelial cells is an important and exciting advance in the field of tissue engineering. Here, we describe recent developments in the use of stem cells for the development of replacement vessels. These paradigm shifts in vascular engineering now offer a new route for effective clinical therapy.     

New advances in stem cell biology: A perspective from gametogenesis

Regeneration of adult tissues relies on a small population of adult stem cells located in a specialized microenvironment. The adult stem cells divide continuously to produce new stem cells, as well as differentiated daughter cells to replenish lost cells due to damage or aging. The molecular mechanisms controlling their ability to divide, self-renew and differentiate remain largely undiscovered. The Drosophila reproductive systems have proven to be excellent models to understand the basic mechanisms regulating stem cell function. This report summarizes some of the recent advances in this field that were presented at the 49(th) Drosophila Research Conference held in San Diego in April 2008.     

A tissue model for the study of cell proliferation parameters in vivo

Summary An intact tissue model which can be used for detailed microscopic studies, quantitative cytochemical analysis and biochemical analysis has been explored, using a number of cell proliferation parameters. The preparations consist of mesenterial windows from rats dispersed on object slides. A technique for determining, in one and the same preparation, DNA content and mitotic activity of individual, selected cell types, and DNA synthesis in terms of incorporation of tritiated thymidine into DNA is described.Supported by grants from the Swedish Medical Research Council (Project 12X-2235) and from the Medical Faculty, University of LinköpingWe thank Brita Söderlund and Iréne Svensson for skilful technical assistance     

Taxus sp. cell, tissue and organ cultures as alternative sources for taxoids production: a literature survey

The literature concerning the tissue culture of Taxus sp. as an alternative source for taxoid production is reviewed. The aim of this review is to summarize and discuss the progress achieved with the approaches and methods used for the establishment of various Taxus culture systems, the methods used for the evaluation of taxoid production, the multiple factors which control taxoid production and the feasibility of the in vitro production of taxoids on a commercial scale.     

A new in vitro model for stem cell differentiation and interaction

Development involves an interplay between various cell types from their birth to their disappearance by differentiation, migration, or death. Analyzing these interactions provides insights into their roles during the formation of a new organism. As a study tool for these interactions, we have created a model based on embryoid bodies (EBs) generated from mouse embryonic stem (mES) cells, which can be used to visualize the differentiation of mES cells into specific cell types while at the same time allowing controlled removal of this same cell population using an enzyme–prodrug approach. Cell-specific expression of Cre induces a switch of EGFP expression to LacZ. Furthermore, it leads to the expression of nitroreductase (NTR), which in combination with the prodrug CB1954 induces apoptosis. Here, we validate this model by showing expression of LacZ and NTR after Cre-mediated recombination. Additionally we show, as an example, that we can target the endothelial cells in EBs using the Tie-2 promoter driving Cre. Ablating Cre-expressing cells by adding CB1954 to the culture led to an abrogated vascular formation. This system can easily be adapted to determine the fate and interaction of many different cell types provided that there is a cell-type-specific promoter available.     

Young and excitable: the function of new neurons in the adult mammalian brain

Adult neurogenesis occurs in most species and is regulated by a wide variety of environmental and pharmacological challenges. The functional integration of neurons generated in the adult was first demonstrated in songbirds more than two decades ago. In the adult mammalian brain, neurons are continuously generated in two structures, the olfactory bulb and the hippocampus. Current evidence suggests that adult-born immature neurons have distinct electrophysiological properties from old neurons, and proposed roles in a variety of functions including olfaction, learning and mood regulation.     

A method for the rapid establishment of normal adult mammalian colonic epithelial cell cultures

Summary Normal colonic epithelial cell cultures of mammalian origin are required to facilitate the study of both normal cellular functions as well as pathogenesis of certain (human) colonic diseases. To date, little information is available regarding the growth requirements of colonic epithelial cells in culture of eitehr animal or human origin. Such data would enable the development of a long-term culture system for these cells. In this study, we present methodology that results in the establishment of homogeneous cultures of adult rabbit colonic epithelial reproducibly, quickly, and in quantity. The epithelial nature of the cultures is unambiguously established by intermediate filament typing using antikeratin antibodies. Such culutres can now be used for a variety of functional studies as well as to investigate the growth requirements of colonic epithelial in culture. This work was supported by the Blinder Foundation for Crohn’s Disease Research, Harbor UCLA IBD Center (AM 36200) and grant AM 27806 from the National Institutes of Health, Bethesda, MD.     

Switching roles: the functional plasticity of adult tissue stem cells

Adult organisms have to adapt to survive, and the same is true for their tissues. Rates and types of cell production must be rapidly and reversibly adjusted to meet tissue demands in response to both local and systemic challenges. Recent work reveals how stem cell (SC) populations meet these requirements by switching between functional states tuned to homoeostasis or regeneration. This plasticity extends to differentiating cells, which are capable of reverting to SCs after injury. The concept of the niche, the micro‐environment that sustains and regulates stem cells, is broadening, with a new appreciation of the role of physical factors and hormonal signals. Here, we review different functions of SCs, the cellular mechanisms that underlie them and the signals that bias the fate of SCs as they switch between roles.     

A subcellular prefractionation protocol for minute amounts of mammalian cell cultures and tissue

Subcellular localization represents an essential, albeit often neglected, aspect of proteome analysis. Generally, the subcellular location of proteins determines the function of cells and tissues. Here we present a robust and versatile prefractionation protocol for mammalian cells and tissues which is appropriate for minute sample amounts. The protocol yields three fractions: a nuclear, a cytoplasmic, and a combined membrane and organelle fraction. The subcellular specificity and the composition of the fractions were demonstrated by immunoblot analysis of five marker proteins and analysis of 43 proteins by two-dimensional gel electrophoresis and mass spectrometry. To cover all protein species, both conventional two-dimensional and benzyldimethyl-n-hexadecyl ammonium chloride-sodium dodecyl sulfate (16-BAC-SDS) gel electrophoresis were performed. Integral membrane proteins and strongly basic nuclear histones were detected only in the 16-BAC-SDS gel electrophoresis system, confirming its usefulness for proteome analysis. All but one protein complied to the respective subcellular composition of the analyzed fractions. Taken together, the data make our subcellular prefractionation protocol an attractive alternative to other prefractionation methods which are based on less physiological protein properties.     

Use of papain in the preparation of adult mammalian skeletal muscle for tissue culture

Summary This report describes in detail a method of enzymatic separation of adult mammalian muscle using papain. The procedure has proved valuable in the preparation of suspensions of muscle cell pieces from normal human skeletal muscle obtained from patients of all ages, from 3 months to 79 years. Muscle cultures have been successfully growth from biopsy material from boys with Duchenne’s muscular dystrophy and from their mothers. The procedure was initially established with adult canine skeletal muscle and has also been used for monkey muscle. Small pieces of skeletal muscle are chopped in a solution of 0.05% papain and 0.01% cysteine hydrochloride in Ca²⁺-and Mg²⁺-free balanced salt solution and transferred in the papain solution to a flask, in which they are incubated at 37°C for 10 min with occasional agitation. The resulting cell suspension is collected and the remaining pieces are treated with further portions of fresh papain until only connective tissue remains. The cell pellets obtained by centrifugation are resuspended in Eagle’s minimum essential medium (supplemented with 20% fetal calf serum) and transferred to culture chambers. The muscle can be observed at all times, during the separation procedure and subsequently in culture. The events occurring during skeletal muscle regeneration can be followed. Using the same papain preparation, myoblasts and myotubes may be subcultured and collected for indefinite frozen storage in dimethylsulfoxide. This work was supported by a grant-in-aid from the American Heart Association with funds contributed in part by the Chicago Heart Association, the Pharmaceutical Manufactures Association, and National Institutes of Health Research Grant NS 10385 from the Institute of Neurological Diseases and Stroke.