Current Pharmaceutical Biotechnology - Volume 8, Issue 3, 2007

In 1913, Santiago Ramon Y Cajal, one of the fathers of neuroscience and a Nobel prize laureate, wrote “... the functional specialization of the brain imposes on the neurons two great lacunae; proliferation inability and irreversibility of intraprotoplasmic differentiation. It is for this reason that, once the development was ended, the founts of growth and regeneration of axons and dendrites dried up irrevocably. In adult centers, the nerve paths are something fixed, ended and immutable. Everything may die, nothing may be regenerated. It is for the science of the future to change, if possible, this harsh decree”. However, today, in what Cajal may have termed the future, neuroscientists know that this ‘harsh decree’ is not as harsh as first thought. Rather, the brain's ability to produce new neurons via neurogenesis in the two developmentally active germinal zones is highly regulated and appears vital for cognition, memory and for the repair and replacement of damaged neurons after injury. Although the first studies showing that progenitor cell proliferation and neurogenesis occur in the mammalian brain were first reported in the 60's, these studies did not receive the attention they deserved and until the 90's, we were on the heel of the learning curve that was about to become exponential. In particular, it was the demonstration by Eriksson et al. in 1998 that neurogenesis occurs in the human hippocampus that first made the field realise that these germinal zone cells might be useful for the treatment of disease in humans. Although neuroscientists tend to focus on the advances in biology, it is very evident that many biological advances occur subsequent to the development of technology. In this edition of JCPB, we will focus on the studies performed and the methods used to analyse progenitor cell populations in vivo and in vitro. Our review series begins by defining the progenitor cell populations and clarifying the potentially confusing nomenclature used in stem and progenitor cell biology. We also review the techniques used to examine the unique cohort of proteins that progenitor cells express, thus making them distinct as a cell population. Then our series examines the pitfalls that have entrapped many, when using double and triple labelling techniques, due to confounding artefacts. The next review describes neurosphere formation as a measure of self-renewal and differentiation capacity of progenitor/stem cells in vitro with comparison to the in vivo situation. Then fluorescence activated cell sorting (FACS) is reviewed as a method for the detection of specific cell populations based on cell surface markers; these techniques appear to be coming of age in the field of neural progenitor cells also. From there, our series focuses on the temporal expression of endogenous cell cycle proteins through the progenitor cell cycle and reveals how the analysis of these proteins may take us ‘beyond BrdU’. We have also included a review of microarrays for high throughput detection of differentially expressed RNA and DNA in different progenitor cell lines. Then, the techniques used and the results reported for analysing progenitor cell migration in vivo and in vitro are reviewed- this detailed review is not to be missed. The final review is focused on progenitor cells from hippocampi donated by patients that undergo temporal lobectomy for intractable epilepsy. This final review hits the core of practical progenitor cell biology; the results reviewed do not present a model or theory but rather an insight into a human neurological disease itself and the effect the disease has on progenitor cells, or vice versa. We hope that you find these reviews both timely and interesting. We also hope that from these reviews you will be inspired with new approaches and ideas for answering the many questions that remain concerning progenitor cells in the brain.

Since the discovery of endogenous progenitor cells in two brain regions in the adult, the notion that progenitor cells might be useful for repairing damaged neurons or replacing dead neurons has gone from fiction to a reality, at least in the laboratory setting. Progenitor cells have the unique ability to be able to produce new neurons in response to endogenous and exogenous cues from their microenvironment in the brain and from the environment of the organism. However, in models of several disorders and insults the regenerative potential of the central nervous system need external enhancing. In this review we begin by focussing on the developments in the field of neurobiology that have led to the specific study of neural progenitor cell biology. In particular we discuss the two germinal niches, the subventricular zone and the subgranular zone, as well as how various neurological diseases affect these niches. We furthermore try to define primary progenitor cell disorders and secondary progenitor cell responses. The second part of this review focuses on proteomic approaches for studying progenitor cells. These techniques allow the array of proteins that are expressed by progenitor cells to be determined and further more allow comparisons between diseased and normal cells or treated and untreated cell populations. If we can induce neural progenitor cells to generate functional neurons in the central nervous system (CNS) then the burden of neurological disorders may be eased in the future. The advances in proteomic technology have and will enable further understanding of the regulatory processes in these cells so that progenitor cell integration and differentiation can be enhanced. Hopefully an increase in knowledge of progenitor cell biology will have a major impact on clinical practice.

Bromodeoxyuridine (BrdU) is widely used for labeling dividing cells to determine their fate. In particular, the analysis of neurogenesis in the adult mammalian brain has made significant progress through the use of this technique. However; when using BrdU for labeling, there are several issues to consider in order to minimalize possible cytotoxicity or false-positive labeling. This current review summarizes methodological and technical aspects of BrdU administration and detection, compares alternative methods and gives recommendations on how to avoid labeling artifacts.

Fluorescence activated cell sorting (FACS) in the field of stem cell biology has become an indispensable tool for defining and separating rare cell populations with a high degree of purity. Steady progress has been made in this regard, but the intrinsic lability of the stem cell phenotype presents a different challenge and there are many technical caveats. FACS remains, however, the technology of choice for reporting and characterizing rare cell populations such as stem cells.

Since their initial description in 1992, neurospheres have appeared in some aspect of more than a thousand published studies. Despite their ubiquitous presence in the scientific literature, there is little consensus regarding the fundamental defining characteristics of neurospheres; thus, there is little agreement about what, if anything, the neurosphere assay can tell us about the relative abundance or behavior of neural stem cells in vivo. In this review we will examine some of the common features of neurospheres, and ask if these features should be interpreted as a proxy for neural stem cells. In addition, we will discuss ways in which the neurosphere assay has been used to evaluate in vivo treatment/manipulation, and will suggest appropriate ways in which neurosphere data should be interpreted, vis-a-vis the neural stem cell. Finally, we will discuss a relatively new in vitro approach, the Neural-Colony Forming Cell Assay, which provides a more meaningful method of quantifying bona fide neural stem cells without conflating them with more growth-restricted progenitor cells.

One of the greatest scientific discoveries of the 20th century is that the mammalian brain can give rise to new neurons throughout the lifespan. The phenomenon of adult neurogenesis raises hopes of harnessing neural stem cell for brain repair, and has sparked interest in novel roles for these new neurons, such as olfaction, spatial memory, and even regulation of mood. Traditionally, studies on adult neurogenesis have relied on exogenous markers of DNA synthesis, such as bromodeoxyuridine (BrdU), to label and track the birth of new cells. However, the exponential increase in our knowledge of endogenous markers of cycling cells has ushered in a new era of stem cell biology. Here we review the strides made in using endogenous cell cycle proteins to study adult neurogenesis in vivo. We (1) discuss the distribution of endogenous cell cycle proteins in proliferative regions of the adult mammalian brain; (2) review cell cycle phase-specific information gained from analyzing a combination of endogenous cell cycle proteins; and (3) provide data on the regulation of cell cycle proteins by a robust inhibitor of proliferation, morphine. The ability of BrdU to birthdate cells ensures it will always serve a role in studies of adult neurogenesis, thus preventing us from moving entirely ‘beyond BrdU’. However, it is hoped that this review will provide interested researchers with the tools needed to apply the powerful and relatively novel approach of analyzing endogenous cell cycle proteins to the study of stem cells in general and adult neurogenesis in particular.

Stem cells represent the key to tissue genesis, regeneration, and turnover. This notion has spawned the concept of regenerative medicine, or stem cell based therapies to supplement degenerating or damaged tissues. However, stem cells may also represent a preferential target of carcinogens. The unique ability of stem cells to self-renew and to differentiate into multiple phenotypes implies that all stem cells share a common transcriptional signature. A better knowledge of the stem cell transcriptome appears to be fundamental to fully achieve the potential of regenerative medicine, and may lead to new strategies for cancer prevention and treatment. Elucidation of the transcriptional programming and molecular mechanisms which direct stem cell self-renewal, differentiation, and tumorigenesis should provide key insights into deciphering exactly how “stemness” is maintained, as well as the molecular basis of cell plasticity and cancer development. cDNA and oligonucleotide microarrays are the most accessible transcriptome profiling methods to date, providing the unique opportunity to compare global gene expression patterns among different cell populations. Microarray technologies have been applied to three major areas of stem cell research: maintenance of pluripotency, development of uniform and regulated differentiation, and microenvironment analyses. The aim of the present review is to summarize state-of-the-art transcriptional profiling of different stem cell lines, cancer stem cells, and the niches these cells occupy in vivo.

One of the most surprising aspects of neural development is that cells do not remain in their birthplace but actively migrate along a variety of routes to their final destinations. This review traces past, present, and future techniques used to analyze progenitor cell migration in the brain, and also discusses their relevant strengths and weaknesses. The large majority of information regarding cell migration is from studies where migratory cells have been labeled, but in which the actual movements are not observed, ie., from static experiments. More recently, dynamic imaging of cell migration in living slices and, even in vivo, has provided a glimpse of how complex these phenomena truly are. A variety of new techniques, such as 2-photon videomicroscopy, are emerging that will continue to add to our body of knowledge concerning the migration of cells in the central nervous system.

Mesial temporal lobe epilepsy (mTLE) is a neurological condition characterized by the occurrence of spontaneous recurrent seizures originating from mesial structures involving the hippocampus within the temporal lobe. This condition is often associated with pathological features in the hippocampus such as neuronal cell loss, widening of the granule cell layer, astrogliosis and mossy fibre spouting. At present, the mechanisms underlying these pathological features are unclear. However, recent advances in adult neurogenesis studies in mTLE animals and patients suggest that newly generated neurons may contribute to the pathogenesis of ongoing epileptogenesis. This article will review the recent animal and human studies on adult neurogenesis in mTLE and discuss how these results suggests that adult endogenous neurogenesis may not always be reparative in the mTLE and may be targeted in new therapeutic strategies for mTLE.