Rodney L. Rietze

Enumeration of neural stem and progenitor cells in the neural colony-forming cell assay.

Advancement in our understanding of the biology of adult stem cells and their therapeutic potential relies heavily on meaningful functional assays that can identify and measure stem cell activity in vivo and in vitro. In the mammalian nervous system, neural stem cells (NSCs) are often studied using a culture system referred to as the neurosphere assay. We previously challenged a central tenet of this assay, that all neurospheres are derived from a NSC, and provided evidence that it overestimates NSC frequency, rendering it inappropriate for quantitation of NSC frequency in relation to NSC regulation. Here we report the development and validation of the neural colony‐forming cell assay (NCFCA), which discriminates stem from progenitor cells on the basis of their proliferative potential. We anticipate that the NCFCA will provide additional clarity in discerning the regulation of NSCs, thereby facilitating further advances in the promising application of NSCs for therapeutic use.

Suppressor of cytokine signaling 2 regulates neuronal differentiation by inhibiting growth hormone signaling

The intracellular mechanisms that determine the response of neural progenitor cells to growth factors and regulate their differentiation into either neurons or astrocytes remain unclear. We found that expression of SOCS2, an intracellular regulator of cytokine signaling, was restricted to mouse progenitor cells and neurons in response to leukemia inhibitory factor (LIF)-like cytokines. Progenitors lacking SOCS2 produced fewer neurons and more astrocytes in vitro, and Socs2−/− mice had fewer neurons and neurogenin-1 (Ngn1)-expressing cells in the developing cortex, whereas overexpression of SOCS2 increased neuronal differentiation. We also report that growth hormone inhibited Ngn1 expression and neuronal production, and this action was blocked by SOCS2 overexpression. These findings indicate that SOCS2 promotes neuronal differentiation by blocking growth hormone–mediated downregulation of Ngn1.

Exercise increases neural stem cell number in a growth hormone-dependent manner, augmenting the regenerative response in aged mice

The exercise‐induced enhancement of learning and memory, and its ability to slow age‐related cognitive decline in humans led us to investigate whether running stimulates periventricular (PVR) neural stem cells (NSCs) in aging mice, thereby augmenting the regenerative capacity of the brain. To establish a benchmark of normal aging on endogenous NSCs, we harvested the PVR from serial vibratome sections through the lateral ventricles of juvenile (6‐8 weeks), 6‐, 12‐, 18‐, and 24‐month‐old mice, culturing the cells in the neural colony‐forming cell assay. A significant decline in NSC frequency was apparent by 6 months (∼40%), ultimately resulting in a ∼90% reduction by 24 months. Concurrent with this decline was a progressive loss in regenerative capacity, as reflected by an incomplete repopulation of neurosphere‐forming cells following gamma cell irradiation‐induced depletion of the PVR. However, voluntary exercise (i.e., 21 days of running) significantly increased NSC frequency in mice ≥ 18 months of age, augmenting the regeneration of irradiation‐ablated periventricular cells and restoring NSC numbers to youthful levels. Importantly, and consistent with the demonstrated ability of growth hormone (GH) to increase NSC proliferation, and the elevated secretion of GH during exercise, exercise failed to stimulate NSCs in GH receptor‐null mice. These findings now provide a novel basis for understanding the ability of exercise to delay the onset and rate of decline in neurodegenerative conditions not typically associated with the hippocampus and suggest that the GH‐dependent activation of endogenous NSCs may be effective in reversing or preventing age‐related neurodegeneration in humans. STEM CELLS 2009;27:2044–2052

The Transcriptional Coactivator Querkopf Controls Adult Neurogenesis

The adult mammalian brain maintains populations of neural stem cells within discrete proliferative zones. Understanding of the molecular mechanisms regulating adult neural stem cell function is limited. Here, we show that MYST family histone acetyltransferase Querkopf (Qkf, Myst4, Morf)-deficient mice have cumulative defects in adult neurogenesis in vivo, resulting in declining numbers of olfactory bulb interneurons, a population of neurons produced in large numbers during adulthood. Qkf-deficient mice have fewer neural stem cells and fewer migrating neuroblasts in the rostral migratory stream. Qkf gene expression is strong in the neurogenic subventricular zone. A population enriched in multipotent cells can be isolated from this region on the basis of Qkf gene expression. Neural stem cells/progenitor cells isolated from Qkf mutant mice exhibited a reduced self-renewal capacity and a reduced ability to produce differentiated neurons. Together, our data show that Qkf is essential for normal adult neurogenesis.

Determination of Somatic and Cancer Stem Cell Self-Renewing Symmetric Division Rate Using Sphere Assays

Representing a renewable source for cell replacement, neural stem cells have received substantial attention in recent years. The neurosphere assay represents a method to detect the presence of neural stem cells, however owing to a deficiency of specific and definitive markers to identify them, their quantification and the rate they expand is still indefinite. Here we propose a mathematical interpretation of the neurosphere assay allowing actual measurement of neural stem cell symmetric division frequency. The algorithm of the modeling demonstrates a direct correlation between the overall cell fold expansion over time measured in the sphere assay and the rate stem cells expand via symmetric division. The model offers a methodology to evaluate specifically the effect of diseases and treatments on neural stem cell activity and function. Not only providing new insights in the evaluation of the kinetic features of neural stem cells, our modeling further contemplates cancer biology as cancer stem-like cells have been suggested to maintain tumor growth as somatic stem cells maintain tissue homeostasis. Indeed, tumor stem cells resistance to therapy makes these cells a necessary target for effective treatment. The neurosphere assay mathematical model presented here allows the assessment of the rate malignant stem-like cells expand via symmetric division and the evaluation of the effects of therapeutics on the self-renewal and proliferative activity of this clinically relevant population that drive tumor growth and recurrence.

Comparative analysis of the frequency and distribution of stem and progenitor cells in the adult mouse brain.

The neurosphere assay can detect and expand neural stem cells (NSCs) and progenitor cells, but it cannot discriminate between these two populations. Given two assays have purported to overcome this shortfall, we performed a comparative analysis of the distribution and frequency of NSCs and progenitor cells detected in 400 μm coronal segments along the ventricular neuraxis of the adult mouse brain using the neurosphere assay, the neural colony forming cell assay (N‐CFCA), and label‐retaining cell (LRC) approach. We observed a large variation in the number of progenitor/stem cells detected in serial sections along the neuraxis, with the number of neurosphere‐forming cells detected in individual 400 μm sections varying from a minimum of eight to a maximum of 891 depending upon the rostral‐caudal coordinate assayed. Moreover, the greatest variability occurred in the rostral portion of the lateral ventricles, thereby explaining the large variation in neurosphere frequency previously reported. Whereas the overall number of neurospheres (3730 ± 276) or colonies (4275 ± 124) we detected along the neuraxis did not differ significantly, LRC numbers were significantly reduced (1186 ± 188, 7 month chase) in comparison to both total colonies and neurospheres. Moreover, approximately two orders of magnitude fewer NSC‐derived colonies (50 ± 10) were detected using the N‐CFCA as compared to LRCs. Given only 5% of the LRCs are cycling (BrdU+/Ki‐67+) or competent to divide (BrdU+/Mcm‐2+), and proliferate upon transfer to culture, it is unclear whether this technique selectively detects endogenous NSCs. Overall, caution should be taken with the interpretation and employment of all these techniques.

Growth hormone responsive neural precursor cells reside within the adult mammalian brain

The detection of growth hormone (GH) and its receptor in germinal regions of the mammalian brain prompted our investigation of GH and its role in the regulation of endogenous neural precursor cell activity. Here we report that the addition of exogenous GH significantly increased the expansion rate in long-term neurosphere cultures derived from wild-type mice, while neurospheres derived from GH null mice exhibited a reduced expansion rate. We also detected a doubling in the frequency of large (i.e. stem cell-derived) colonies for up to 120 days following a 7-day intracerebroventricular infusion of GH suggesting the activation of endogenous stem cells. Moreover, gamma irradiation induced the ablation of normally quiescent stem cells in GH-infused mice, resulting in a decline in olfactory bulb neurogenesis. These results suggest that GH activates populations of resident stem and progenitor cells, and therefore may represent a novel therapeutic target for age-related neurodegeneration and associated cognitive decline.

Oncostatin M regulates neural precursor activity in the adult brain

The regulation of neural precursor cell (NPC) activity is the major determinant of the rate of neuronal production in neurogenic regions of the adult brain. Here, we show that Oncostatin M (Osm) and its receptor, OsmRβ, are both expressed in the subventricular zone (SVZ) and that in contradistinction to leukemia inhibitory factor and ciliary neutrophic factor, Osm directly inhibits the proliferation of adult NPCs as measured by a decreased level of neurosphere formation in vitro. Similarly, intraventricular infusion of Osm dramatically decreases the pool of NPCs in both the SVZ and the hippocampus. In keeping with the inhibitory action of Osm, we reveal that mice lacking OsmRβ have substantially more NPCs in the SVZ, the hippocampus and the olfactory bulb, demonstrating that endogenous Osm signaling is important for NPC homeostasis. Finally, we show that Osm can also inhibit clonal growth of glioblastoma‐derived neurospheres, further supporting the close link between NPCs and tumor stem cells.

Oligodendrocytes from neural stem cells express alpha-synuclein: increased numbers from presenilin 1 deficient mice.

&agr;-Synuclein normally a synaptic vesicle-associated cytoplasmic protein is the major component of filamentous inclusions of neurons in Parkinsons disease and dementia with Lewy bodies. It is also the major component of glial inclusions of multiple system atrophy. In characterizing cells derived from embryonic neural stem cells we found all oligodendrocytes had strong cytoplasmic expression of &agr;-synuclein. Comparison of cells from presenilin 1 (PS1)-deficient mice with wild type revealed a 7-fold increase in oligodendrocytes. Western blotting analysis indicated the cells contained &agr;-synuclein monomers and SDS-stable dimers and trimers. This cell system of oligodendroglial &agr;-synuclein expression is a useful system to study &agr;-synuclein metabolism in the cell type affected in multiple system atrophy. Increased oligodendroglial cell numbers from PS1-deficient cells provides further evidence for a role of PS1-dependent Notch signalling in cell fate decisions.

The neurosphere assay, a method under scrutiny

Objectives: The aim of this review is to provide an overview of the fundamental features of the neurosphere assay (NSA), which was initially described in 1992, and has since been used not only to detect the presence of stem cells in embryonic and adult mammalian neural tissues, but also to study their characteristics in vitro. Implicit in this review is a detailed examination of the limitations of the NSA, and how this assay is most accurately and appropriately used. Finally we will point out criteria that should be challenged to design alternative ways to overcome the limits of this assay. Methods: NSA is used to isolate putative neural stem cells (NSCs) from the central nervous system (CNS) and to demonstrate the critical stem cell attributes of proliferation, extensive self-renewal and the ability to give rise to a large number of differentiated and functional progeny. Nevertheless, the capability of neural progenitor cells to form neurospheres precludes its utilisation to accurately quantify bona fide stem cell frequency based simply on neurosphere numbers. New culture conditions are needed to be able to distinguish the activity of progenitor cells from stem cells. Conclusion: A commonly used, and arguably misused, methodology, the NSA has provided a wealth of information on precursor activity of cells derived from the embryonic through to the aged CNS. Importantly, the NSA has contributed to the demise of the ‘no new neurogenesis’ dogma, and the beginning of a new era of CNS regenerative medicine. Nevertheless, the interpretations arising from the utilisation of the NSA need to take into consideration its limits, so as not to be used beyond its specificity and sensitivity.