Developing the control criterion for continuous culture of microorganisms

A short survey and critical analysis of previously proposed criteria for growth control of populations of microorganisms in the chemostat are presented. Based on the analysis of a mathematical model of the steady-state of a microbial population in the chemostat, an adequate control criterion is suggested, along with a method to identify the corresponding regulating factors. The new control criterion is expressed as a product of the factor transformation coefficient and the biomass sensitivity coefficient (SC) with respect to the change of the factor at the chemostat inlet (referred to in the sequel as the biomass SC). The control criterion determines the strength of the control exerted by this or that factor. The method of determination of the regulating factors consists in experimental determination of the real SCs for factors and the biomass and in calculating on this basis the corresponding ideal SCs for constant factor transformation coefficients. The ideal SCs are shown to add up to an integer value, a constraint that we call "quantization" relationships. Such relationships are used to test the completeness of the drawn list of control factors. The proposed method was applied to our own and literature data.     

Efficient generation of functional Schwann cells from adipose-derived stem cells in defined conditions

Schwann cells (SCs) are hitherto regarded as the most promising candidates for viable cell-based therapy to peripheral nervous system (PNS) injuries or degenerative diseases. However, the extreme drawbacks of transplanting autologous SCs for clinical applications still represent a significant bottleneck in neural regenerative medicine, mainly owing to the need of sacrificing a functional nerve to generate autologous SCs and the nature of slow expansion of the SCs. Thus, it is of great importance to establish an alternative cell system for the generation of sufficient SCs. Here, we demonstrated that adipose-derived stem cells (ADSCs) of rat robustly give rise to morphological, phenotypic and functional SCs using an optimized protocol. After undergoing a 3-week in vitro differentiation, almost all of treated ADSCs exhibited spindle shaped morphology similar to genuine SCs and expressed SC markers GFAP and S100. Most importantly, apart from acquisition of SC antigenic and biochemical features, the ADSC-derived SCs were functionally identical to native SCs as they possess a potential ability to form myelin, and secret nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glia-derived neurotrophic factor (GDNF). The current study may provide an ideal strategy for harvesting sufficient SCs for cell-based treatment of various peripheral nerve injuries or disorders.     

Contributions of <Emphasis Type="Italic">I</Emphasis><Subscript>h</Subscript> to feature selectivity in layer II stellate cells of the entorhinal cortex

Stellate cells (SCs) of the entorhinal cortex generate prominent subthreshold oscillations that are believed to be important contributors to the hippocampal theta rhythm. The slow inward rectifier I _h is expressed prominently in SCs and has been suggested to be a dominant factor in their integrative properties. We studied the input-output relationships in stellate cells (SCs) of the entorhinal cortex, both in control conditions and in the presence of the I _h antagonist ZD7288. Our results show that I _h is responsible for SCs’ subthreshold resonance, and contributes to enhanced spiking reliability to theta-rich stimuli. However, SCs still exhibit other traits of rhythmicity, such as subthreshold oscillations, under I _h blockade. To clarify the effects of I _h on SC spiking, we used a generalized form of principal component analysis to show that SCs select particular features with relevant temporal signatures from stimuli. The spike-selected mix of those features varies with the frequency content of the stimulus, emphasizing the inherent nonlinearity of SC responses. A number of controls confirmed that this selectivity represents a stimulus-induced change in the cellular input-output relationship rather than an artifact of the analysis technique. Sensitivity to slow features remained statistically significant in ZD7288. However, with I _h blocked, slow stimulus features were less predictive of spikes and spikes conveyed less information about the stimulus over long time scales. Together, these results suggest that I _h is an important contributor to the input-output relationships expressed by SCs, but that other factors in SCs also contribute to subthreshold oscillations and nonlinear selectivity to slow features. Action Editor: Xiao-Jing Wang     

Effect of panaxydol on hypoxia-induced cell death and expression and secretion of neurotrophic factors (NTFs) in hypoxic primary cultured Schwann cells

It has been shown that panaxydol (PND) can mimic the neurotrophic effect of nerve growth factor (NGF) normally secreted by Schwann cells (SC) and protect neurons against injury. To evaluate the effect of PND on hypoxia-induced SC death and expression and secretion of neurotrophic factors (NGF and brain derived neurotrophic factor (BDNF)), hypoxic SCs were cultured in vitro and then treated with PND (0-20 microM). The MTT (3(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, immunocytochemistry, ELISA and RT-PCR were employed to examine the effects. We found that hypoxia resulted in a significant decrease in SCs viability (MTT: 64+/-4.7% of control group) and nearly a 3.3-fold increase of intracellular level of active caspase-3. PND (5-20 microM) treatment significantly rescued the SCs from hypoxia-induced injury (85+/-8.2%; 92+/-8.6%; 87+/-7.3%) and reduced caspase-3 activity with the maximal effect occurred at 10 microM (P<0.01), reducing to about 1.6-fold of control level. Furthermore, PND treatment also enhanced NGF and BDNF mRNA levels in hypoxic SCs and promoted protein expression and secretion. BDNF mRNA in hypoxic SCs was restored to about 90% of normal level and NGF mRNA was elevated to 1.4-fold of control after 10 microM PND treatment. These observations showed that PND protects primary cultured SCs against hypoxia-induced injury and enhances NTF-associated activities.     

Synaptonemal complex karyotyping in spermatocytes of the Chinese hamster (Cricetulus griseus)

Relative length is a constant and distinctive characteristic for each autosomal SC, despite variations in absolute length from cell to cell. Arm ratio is distinctive for each SC except for two of the three sub-acrocentrics, and serves, together with relative length, for identification. The constancy of relative length and arm ratios indicates biological stability and lack of physical distortion in these spread preparations. There is a 11 relationship between relative lengths of autosomal SCs and mitotic autosomes; their arm ratios are also similar. These close parallels provide strikingly similar SC and somatic karyotypes. Variability was observed in sub-acrocentric arm ratios and in lengths of unpaired X and Y axes, correlated with the presence of constitutive heterochromatin. — Utilizing progressive differentiations of the X and Y chromosomes for staging, it is demonstrated that autosomal SCs decrease in length from late zygotene to mid-pachytene, and then increase at late pachytene. Within a nucleus, synchrony of length changes is maintained. It is concluded that the factors governing autosomal SC length are regular for any given bivalent from cell to cell, and may be related to those that control somatic autosome length relationships. — The X and Y axes differ quantitatively as well as qualitatively from autosomal SCs. The SC portion of the X and Y is constant in length through most of pachytene; the unpaired axes shorten and lengthen, but not in proportion to autosomal SCs. X and Y relative lengths and arm ratios vary throughout pachytene and do not maintain proportionality with somatic values. The evidence suggests, but does not prove, that the long arm of the X is paired with the short arm of the Y. — Twists occur in autosomal SCs at increasing frequencies throughout pachytene but cannot account for length changes. The number of twists per SC is directly proportional to SC length. Intertwining of SCs is random and proportional to SC length. End-to-end associations of autosomal SCs appear to be random; however, the ends of the X and Y are less often involved in such connections. — The length of axial material in all chromosomes at pachytene, expressed as an equivalent length of DNA double helix, represents 0.013% of the diploid DNA complement.     

Novel method to obtain highly enriched cultures of adult rat Schwann cells

Schwann cells (SCs) can be used to repair both the peripheral and central nervous systems. Therefore, establishment of a procedure to obtain activated, highly proliferative SCs, in an appropriate time for clinical applications, is a prerequisite. Purification is complicated by contamination with fibroblasts which often become the predominant cell type in an in vitro SC culture. This study describes a novel and efficient method to enrich SCs by utilizing the differential detachment properties of the two cell types. In culture, cells were treated with two different media and the chelator, EGTA, which detached SCs faster than fibroblasts and allowed for easy isolation of SCs. Within seven days, high yields of SCs with a purity of greater than 99% were achieved. This was confirmed by immunostaining characterization and flow-cytometric analyses using an antibody against the p75 low affinity nerve growth factor receptor (p75LNGFR). The entire procedure was completed in approximately 21 days. This method has the advantage of being technically easier, faster, and more efficient than other previously described methods. An SC culture that was about 99% homogenous was achieved.     

An improved method for isolating Schwann cells from postnatal rat sciatic nerves

The major difficulty in Schwann cell (SC) purification is contamination by fibroblasts, which usually become the predominant cell type during SC enrichment in vitro. Current reported measures are mainly limited by either high cost or complicated procedures with low cell yields or purity. Our objectives have been to develop an efficient, easily applicable, rapid method to obtain highly purified SC from the sciatic nerve of newborn rats. The method involves two rounds of purification to eliminate fibroblasts with the novel combined use of cytosine-B-arabinoside hydrochloride (Ara-C) action and differential cell detachment. Cultured cells were first treated with Ara-C for 24 h. The medium was replaced with the growth medium containing 20 ng/ml human heregulin1-β1 extracellular domain (HRG1-β1 ECD). After another 48 h in culture, the cells were treated with 0.05% trypsin, following which SCs, but not fibroblasts, were easily detached from the dishes. The advantage of this method is that the two steps can eliminate the fibroblasts complementarily. Ara-C eliminates most of the fibroblasts growing among SCs, whereas the differential cell detachment technique removes the remainder growing under or interacting with the SC layer. A purity of more than 99% SCs has been obtained, as confirmed by cell morphology and immunostaining. The purified SCs have a spindle-shaped, bipolar, and sometimes tripolar morphology, align in fascicles, and express S-100. The whole procedure takes about 10 days from primary culture to the purified SCs growing to confluence (only half the time reported previously). This protocol provides an alternative method for investigating peripheral nerve regeneration and potentially could be used to produce enough SCs to construct artificial nerve scaffolds in vitro. This work was supported by Tsinghua-Yue-Yuen Medical Sciences Fund, the National Natural Science Foundation of China (contract grant numbers: 30670528, 30700848, 30772443), Beijing Municipal Science & Technology Commission (BMSTC, contract grant number: H060920050430), National Basic Research Program of China (also called the 973 Program, contract grant number: 2005CB623905), and the National Natural Science Foundation of Beijing (contract grant number: 7082090).     

Analyzing Murine Schwann Cell Development Along Growing Axons

The development of peripheral nerves is an intriguing process. Neurons send out axons to innervate specific targets, which in humans are often more than 100 cm away from the soma of the neuron. Neuronal survival during development depends on target-derived growth factors but also on the support of Schwann cells (SCs). To this end SC ensheath axons from the region of the neuronal soma (or the transition from central to peripheral nervous system) to the synapse or neuromuscular junction. Schwann cells are derivatives of the neural crest and migrate as precursors along emerging axons until the entire axon is covered with SCs. This shows the importance of SC migration for the development of the peripheral nervous system and underlines the necessity to investigate this process. In order to analyze SC development, a setup is needed which next to the SCs also includes their physiological substrate for migration, the axon. Due to intrauterine development in vivo time-lapse imaging, however, is not feasible in placental vertebrates like mouse (mus musculus). To circumvent this, we adapted the superior cervical ganglion (SCG) explant technique. Upon treatment with nerve growth factor (NGF) SCG explants extend axons, followed by SC precursors migrating along the axons from the ganglion to the periphery. The beauty of this system is that the SC are derived from a pool of endogenous SC and that they migrate along their own physiological axons which are growing at the same time. This system is especially intriguing, because the SC development along axons can be analyzed by time-lapse imaging, opening further possibilities to gain insights into SC migration.     

The role of the microenvironment on the fate of adult stem cells

Adult stem cells (SCs) exist in all tissues that promote tissue growth, regeneration, and healing throughout life. The SC niche in which they reside provides signals that direct them to proliferate, differentiate, or remain dormant; these factors include neighboring cells, the extracellular matrix, soluble molecules, and physical stimuli. In disease and aging states, stable or transitory changes in the microenvironment can directly cause SC activation or inhibition in tissue healing as well as functional regulation. Here, we discuss the microenvironmental regulation of the behavior of SC and focus on plasticity approaches by which various environmental factors can enhance the function of SCs and more effectively direct the fate of SCs.     

Environmental control of lineage plasticity and stem cell memory

Tissue-resident stem cells (SCs) are critical players in the maintenance of tissue homeostasis. SCs reside in complex and uniquely anatomically organized microenvironments (SC niches), that carefully control SC lineage outputs depending on localized tissue needs. Upon environmental perturbations and tissue stressors, SCs respond and restore the tissue to homeostasis, as well as protect it from secondary assaults. Critical to this function are two key processes, SC lineage plasticity and SC memory. In this review, we delineate the multifactorial determinants and key principles underlining these two remarkable SC behaviors. Understanding lineage plasticity and SC memory will be critical not only to design new regenerative therapies but also to determine how these processes are altered in a multitude of pathologies such as cancer and chronic tissue damage.     

Stem-cell approaches for kidney repair: choosing the right cells

With the increasing rate of end-stage renal failure and limited alternatives for its treatment, stem cell (SC) therapy for kidney injury is urgently needed. Choosing the right SC type is the critical step in realizing the potential of this therapeutic approach. Four possible sources of SCs are envisioned for the development of this type of treatment: (i) bone-marrow-derived SCs (BMSCs), (ii) renal adult SCs, (iii) embryonic SCs and (iv) fetal renal SCs. We suggest that resident SCs recently identified in the Bowman's capsule of adult human kidneys might prospectively be the ideal cell type for treatment of both acute and chronic renal injury because they display the potential to differentiate into multiple types of renal cells. However, BMSCs also represent an attractive alternative, especially for the treatment of patients affected by acute renal failure.     

Naked Mole-Rat is Sensitive to Social Hierarchy Encoded in Antiphonal Vocalization

The maintenance of social relationships is critical for group-dwelling species. Social animals often exhibit behaviors such as antiphonal vocalizations that reduce conflict and maintain affiliations. Naked mole-rats ( Heterocephalus glaber ) have a complex hierarchical society comparable to that of bees and ants. They are also known for their extensive vocal repertoire, which may have evolved in the absence of visual cues. The most frequent vocalization used by naked mole-rats is the soft chirp (SC). It has an antiphonal nature and may function in rank identification and in maintaining affiliations. Relative body weight differences, which are directly related to social rank, are positively correlated with SC emission rates. SCs are elicited from either physical touch or the SC of another conspecific, and other cues might contribute to SC utterance. In the current study, we examined whether an SC alone was able to elicit SC responses. Specifically, we presented artificial SC-like sounds and determined whether the response rate was modulated by the acoustic properties of the stimulus. An analysis of response latency revealed that animals responded to the audio stimuli, and a single audio stimulus could elicit responses from two animals. Thus, antiphony in naked mole-rats may occur among three or more animals. We also found that animals were able to discriminate the acoustic properties of the stimulus and responded more frequently to audio stimuli resembling SCs from large animals than to those resembling SCs from small animals. Therefore, naked mole-rats may be able to judge social relationships (dominant or subordinate) based solely on SCs. The constraints of subterranean habitats and increased social complexity may have led to the evolution of this communication system.     

New multiple labelling method for improved satellite cell identification in human muscle: application to a cohort of power-lifters and sedentary men

Presently applied methods to identify and quantify human satellite cells (SCs) give discrepant results. We introduce a new immunofluorescence method that simultaneously monitors two SC markers (NCAM and Pax7), the basal lamina and nuclei. Biopsies from power-lifters, power-lifters using anabolic substances and untrained subjects were re-examined. Significantly different results from those with staining for NCAM and nuclei were observed. There were three subtypes of SCs; NCAM⁺/Pax7⁺ (94%), NCAM⁺/Pax7⁻ (4%) and NCAM⁻/Pax7⁺ (1%) but large individual variability existed. The proportion of SCs per nuclei within the basal lamina of myofibres (SC/N) was similar for all groups reflecting a balance between the number of SCs and myonuclei to maintain homeostasis. We emphasise that it is important to quantify both SC/N and the number of SCs per fibre. Our multiple marker method is more reliable for SC identification and quantification and can be used to evaluate other markers of muscle progenitor cells.