The mammalian efferent vestibular system utilizes cholinergic mechanisms to excite primary vestibular afferents.

Electrical stimulation of the mammalian efferent vestibular system (EVS) predominantly excites primary vestibular afferents along two distinct time scales. Although roles for acetylcholine (ACh) have been demonstrated in other vertebrates, synaptic mechanisms underlying mammalian EVS actions are not well-characterized. To determine if activation of ACh receptors account for efferent-mediated afferent excitation in mammals, we recorded afferent activity from the superior vestibular nerve of anesthetized C57BL/6 mice while stimulating EVS neurons in the brainstem, before and after administration of cholinergic antagonists. Using a normalized coefficient of variation (CV*), we broadly classified vestibular afferents as regularly- (CV* < 0.1) or irregularly-discharging (CV* > 0.1) and characterized their responses to midline or ipsilateral EVS stimulation. Afferent responses to efferent stimulation were predominantly excitatory, grew in amplitude with increasing CV*, and consisted of fast and slow components that could be identified by differences in rise time and post-stimulus duration. Both efferent-mediated excitatory components were larger in irregular afferents with ipsilateral EVS stimulation. Our pharmacological data show, for the first time in mammals, that muscarinic AChR antagonists block efferent-mediated slow excitation whereas the nicotinic AChR antagonist DHßE selectively blocks efferent-mediated fast excitation, while leaving the efferent-mediated slow component intact. These data confirm that mammalian EVS actions are predominantly cholinergic.

Loss of α-Calcitonin Gene-Related Peptide (αCGRP) Reduces Otolith Activation Timing Dynamics and Impairs Balance.

Calcitonin gene-related peptide (CGRP) is a neuroactive peptide that is thought to play a role at efferent synapses in hair cell organs including the cochlea, lateral line, and semicircular canal. The deletion of CGRP in transgenic mice is associated with a significant reduction in suprathreshold cochlear nerve activity and vestibulo-ocular reflex (VOR) gain efficacy when compared to littermate controls. Here we asked whether the loss of CGRP also influences otolithic end organ function and contributes to balance impairments. Immunostaining for CGRP was absent in the otolithic end organs of αCGRP null (-/-) mice while choline acetyltransferase (ChAT) immunolabeling appeared unchanged suggesting the overall gross development of efferent innervation in otolithic organs was unaltered. Otolithic function was assessed by quantifying the thresholds, suprathreshold amplitudes, and latencies of vestibular sensory-evoked potentials (VsEPs) while general balance function was assessed using a modified rotarod assay. The loss of αCGRP in null (-/-) mice was associated with: (1) shorter VsEP latencies without a concomitant change in amplitude or thresholds, and (2) deficits in the rotarod balance assay. Our findings show that CGRP loss results in faster otolith afferent activation timing, suggesting that the CGRP component of the efferent vestibular system (EVS) also plays a role in otolithic organ dynamics, which when coupled with reduced VOR gain efficacy, impairs balance.

ACh-induced hyperpolarization and decreased resistance in mammalian type II vestibular hair cells.

In the mammalian vestibular periphery, electrical activation of the efferent vestibular system (EVS) has two effects on afferent activity: 1) it increases background afferent discharge and 2) decreases afferent sensitivity to rotational stimuli. Although the cellular mechanisms underlying these two contrasting afferent responses remain obscure, we postulated that the reduction in afferent sensitivity was attributed, in part, to the activation of α9- containing nicotinic acetylcholine (ACh) receptors (α9*nAChRs) and small-conductance potassium channels (SK) in vestibular type II hair cells, as demonstrated in the peripheral vestibular system of other vertebrates. To test this hypothesis, we examined the effects of the predominant EVS neurotransmitter ACh on vestibular type II hair cells from wild-type (wt) and α9-subunit nAChR knockout (α9-/-) mice. Immunostaining for choline acetyltransferase revealed there were no obvious gross morphological differences in the peripheral EVS innervation among any of these strains. ACh application onto wt type II hair cells, at resting potentials, produced a fast inward current followed by a slower outward current, resulting in membrane hyperpolarization and decreased membrane resistance. Hyperpolarization and decreased resistance were due to gating of SK channels. Consistent with activation of α9*nAChRs and SK channels, these ACh-sensitive currents were antagonized by the α9*nAChR blocker strychnine and SK blockers apamin and tamapin. Type II hair cells from α9-/- mice, however, failed to respond to ACh at all. These results confirm the critical importance of α9nAChRs in efferent modulation of mammalian type II vestibular hair cells. Application of exogenous ACh reduces electrical impedance, thereby decreasing type II hair cell sensitivity. NEW & NOTEWORTHY Expression of α9 nicotinic subunit was crucial for fast cholinergic modulation of mammalian vestibular type II hair cells. These findings show a multifaceted efferent mechanism for altering hair cell membrane potential and decreasing membrane resistance that should reduce sensitivity to hair bundle displacements.

Confirming a Role for α9nAChRs and SK Potassium Channels in Type II Hair Cells of the Turtle Posterior Crista.

In turtle posterior cristae, cholinergic vestibular efferent neurons (VENs) synapse on type II hair cells, bouton afferents innervating type II hair cells, and afferent calyces innervating type I hair cells. Electrical stimulation of VENs releases acetylcholine (ACh) at these synapses to exert diverse effects on afferent background discharge including rapid inhibition of bouton afferents and excitation of calyx-bearing afferents. Efferent-mediated inhibition is most pronounced in bouton afferents innervating type II hair cells near the torus, but becomes progressively smaller and briefer when moving longitudinally through the crista toward afferents innervating the planum. Sharp-electrode recordings have inferred that efferent-mediated inhibition of bouton afferents requires the sequential activation of alpha9-containing nicotinic ACh receptors (α9*nAChRs) and small-conductance, calcium-dependent potassium channels (SK) in type II hair cells. Gradations in the strength of efferent-mediated inhibition across the crista likely reflect variations in α9*nAChRs and/or SK activation in type II hair cells from those different regions. However, in turtle cristae, neither inference has been confirmed with direct recordings from type II hair cells. To address these gaps, we performed whole-cell, patch-clamp recordings from type II hair cells within a split-epithelial preparation of the turtle posterior crista. Here, we can easily visualize and record hair cells while maintaining their native location within the neuroepithelium. Consistent with α9*nAChR/SK activation, ACh-sensitive currents in type II hair cells were inward at hyperpolarizing potentials but reversed near -90 mV to produce outward currents that typically peaked around -20 mV. ACh-sensitive currents were largest in torus hair cells but absent from hair cells near the planum. In current clamp recordings under zero-current conditions, ACh robustly hyperpolarized type II hair cells. ACh-sensitive responses were reversibly blocked by the α9nAChR antagonists ICS, strychnine, and methyllycaconitine as well as the SK antagonists apamin and UCL1684. Intact efferent terminals in the split-epithelial preparation spontaneously released ACh that also activated α9*nAChRs/SK in type II hair cells. These release events were accelerated with high-potassium external solution and all events were blocked by strychnine, ICS, methyllycaconitine, and apamin. These findings provide direct evidence that activation of α9*nAChR/SK in turtle type II hair cells underlies efferent-mediated inhibition of bouton afferents.

Muscarinic Acetylcholine Receptors and M-Currents Underlie Efferent-Mediated Slow Excitation in Calyx-Bearing Vestibular Afferents.

Stimulation of vestibular efferent neurons excites calyx and dimorphic (CD) afferents. This excitation consists of fast and slow components that differ >100-fold in activation kinetics and response duration. In the turtle, efferent-mediated fast excitation arises in CD afferents when the predominant efferent neurotransmitter acetylcholine (ACh) activates calyceal nicotinic ACh receptors (nAChRs); however, it is unclear whether the accompanying efferent-mediated slow excitation is also attributed to cholinergic mechanisms. To identify synaptic processes underlying efferent-mediated slow excitation, we recorded from CD afferents innervating the turtle posterior crista during electrical stimulation of efferent neurons, in combination with pharmacological probes and mechanical stimulation. Efferent-mediated slow excitation was unaffected by nAChR compounds that block efferent-mediated fast excitation, but were mimicked by muscarine and antagonized by atropine, indicating that it requires ACh and muscarinic ACh receptor (mAChR) activation. Efferent-mediated slow excitation or muscarine application enhanced the sensitivity of CD afferents to mechanical stimulation, suggesting that mAChR activation increases afferent input impedance by closing calyceal potassium channels. These observations were consistent with suppression of a muscarinic-sensitive K+-current, or M-current. Immunohistochemistry for putative M-current candidates suggested that turtle CD afferents express KCNQ3, KCNQ4, and ERG1-3 potassium channel subunits. KCNQ channels were favored as application of the selective antagonist XE991 mimicked and occluded efferent-mediated slow excitation in CD afferents. These data highlight an efferent-mediated mechanism for enhancing afferent sensitivity. They further suggest that the clinical effectiveness of mAChR antagonists in treating balance disorders may also target synaptic mechanisms in the vestibular periphery, and that KCNQ channel modulators might offer similar therapeutic value.SIGNIFICANCE STATEMENT Targeting the efferent vestibular system (EVS) pharmacologically might prove useful in ameliorating some forms of vestibular dysfunction by modifying ongoing primary vestibular input. EVS activation engages several kinetically distinct synaptic processes that profoundly alter the discharge rate and sensitivity of first-order vestibular neurons. Efferent-mediated slow excitation of vestibular afferents is of considerable interest given its ability to elevate afferent activity over an extended time course. We demonstrate for the first time that efferent-mediated slow excitation of vestibular afferents is mediated by muscarinic acetylcholine receptor (mAChR) activation and the subsequent closure of KCNQ potassium channels. The clinical effectiveness of some anti-mAChR drugs in treating motion sickness suggest that we may, in fact, already be targeting the peripheral EVS.

Maturation of suprathreshold auditory nerve activity involves cochlear CGRP-receptor complex formation.

In adult animals, the neuropeptide calcitonin gene-related peptide (CGRP) is contained in cochlear efferent fibers projecting out to the cochlea, and contributes to increased suprathreshold sound-evoked activity in the adult auditory nerve. Similarly, CGRP applied to the lateral-line organ (hair cell organ) increases afferent nerve activity in adult frogs (post-metamorphic day 30), yet this increase is developmentally delayed from post-metamorphic day 4-30. In this study, we discovered that there was also a developmental delay in increased suprathreshold sound-evoked activity auditory nerve between juvenile and adult mice similar to what had been observed previously in frog. Moreover, juvenile mice with a targeted deletion of the αCGRP gene [CGRP null (-/-)] did not show a similar developmental increase in nerve activity, suggesting CGRP signaling is involved. This developmental delay is not due to a delay in CGRP expression, but instead is due to a delay in receptor formation. We observed that the increase in sound-evoked nerve activity is correlated with increased formation of cochlear CGRP receptors, which require three complexed proteins (CLR, RAMP1, RCP) to be functional. CGRP receptor formation in the cochlea was incomplete at 1 month of age (juvenile), but complete by 3 months (adult), which corresponded to the onset of suprathreshold enhancement of sound-evoked activity in wild-type animals. Taken together, these data support a model for cochlear function that is enhanced by maturation of CGRP receptor complexes.

Pharmacologically distinct nicotinic acetylcholine receptors drive efferent-mediated excitation in calyx-bearing vestibular afferents.

Electrical stimulation of vestibular efferent neurons rapidly excites the resting discharge of calyx/dimorphic (CD) afferents. In turtle, this excitation arises when acetylcholine (ACh), released from efferent terminals, directly depolarizes calyceal endings by activating nicotinic ACh receptors (nAChRs). Although molecular biological data from the peripheral vestibular system implicate most of the known nAChR subunits, specific information about those contributing to efferent-mediated excitation of CD afferents is lacking. We sought to identify the nAChR subunits that underlie the rapid excitation of CD afferents and whether they differ from α9α10 nAChRs on type II hair cells that drive efferent-mediated inhibition in adjacent bouton afferents. We recorded from CD and bouton afferents innervating the turtle posterior crista during electrical stimulation of vestibular efferents while applying several subtype-selective nAChR agonists and antagonists. The α9α10 nAChR antagonists, α-bungarotoxin and α-conotoxin RgIA, blocked efferent-mediated inhibition in bouton afferents while leaving efferent-mediated excitation in CD units largely intact. Conversely, 5-iodo-A-85380, sazetidine-A, varenicline, α-conotoxin MII, and bPiDDB (N,N-dodecane-1,12-diyl-bis-3-picolinium dibromide) blocked efferent-mediated excitation in CD afferents without affecting efferent-mediated inhibition in bouton afferents. This pharmacological profile suggested that calyceal nAChRs contain α6 and ß2, but not α9, nAChR subunits. Selective blockade of efferent-mediated excitation in CD afferents distinguished dimorphic from calyx afferents by revealing type II hair cell input. Dimorphic afferents differed in having higher mean discharge rates and a mean efferent-mediated excitation that was smaller in amplitude yet longer in duration. Molecular biological data demonstrated the expression of α9 in turtle hair cells and α4 and ß2 in associated vestibular ganglia.

Efferent innervation of turtle semicircular canal cristae: comparisons with bird and mouse.

In the vestibular periphery of nearly every vertebrate, cholinergic vestibular efferent neurons give rise to numerous presynaptic varicosities that target hair cells and afferent processes in the sensory neuroepithelium. Although pharmacological studies have described the postsynaptic actions of vestibular efferent stimulation in several species, characterization of efferent innervation patterns and the relative distribution of efferent varicosities among hair cells and afferents are also integral to understanding how efferent synapses operate. Vestibular efferent markers, however, have not been well characterized in the turtle, one of the animal models used by our laboratory. Here we sought to identify reliable efferent neuronal markers in the vestibular periphery of turtle, to use these markers to understand how efferent synapses are organized, and to compare efferent neuronal labeling patterns in turtle with two other amniotes using some of the same markers. Efferent fibers and varicosities were visualized in the semicircular canal of red-eared turtles (Trachemys scripta elegans), zebra finches (Taeniopygia guttata), and mice (Mus musculus) utilizing fluorescent immunohistochemistry with antibodies against choline acetyltransferase (ChAT). Vestibular hair cells and afferents were counterstained using antibodies to myosin VIIa and calretinin. In all species, ChAT labeled a population of small diameter fibers giving rise to numerous spherical varicosities abutting type II hair cells and afferent processes. That these ChAT-positive varicosities represent presynaptic release sites were demonstrated by colabeling with antibodies against the synaptic vesicle proteins synapsin I, SV2, or syntaxin and the neuropeptide calcitonin gene-related peptide. Comparisons of efferent innervation patterns among the three species are discussed.

Loss of α-calcitonin gene-related peptide (αCGRP) reduces the efficacy of the Vestibulo-ocular Reflex (VOR).

The neuroactive peptide calcitonin-gene related peptide (CGRP) is known to act at efferent synapses and their targets in hair cell organs, including the cochlea and lateral line. CGRP is also expressed in vestibular efferent neurons as well as a number of central vestibular neurons. Although CGRP-null (-/-) mice demonstrate a significant reduction in cochlear nerve sound-evoked activity compared with wild-type mice, it is unknown whether and how the loss of CGRP influence vestibular system function. Vestibular function was assessed by quantifying the vestibulo-ocular reflex (VOR) in alert mice. The loss of CGRP in (-/-) mice was associated with a reduction of the VOR gain of ≈50% without a concomitant change in phase. Using immunohistochemistry, we confirmed that, although CGRP staining was absent in the vestibular end-organs of null (-/-) mice, cholinergic staining appeared normal, suggesting that the overall gross development of vestibular efferent innervation was unaltered. We further confirmed that the observed deficit in vestibular function of null (-/-) mice was not the result of nontargeted effects at the level of the extraocular motor neurons and/or their innervation of extraocular muscles. Analysis of the relationship between vestibular quick phase amplitude and peak velocity revealed that extraocular motor function was unchanged, and immunohistochemistry revealed no abnormalities in motor endplates. Together, our findings show that the neurotransmitter CGRP plays a key role in ensuring VOR efficacy.

A review of synaptic mechanisms of vestibular efferent signaling in turtles: extrapolation to efferent actions in mammals.

The vestibular labyrinth of nearly every vertebrate class receives a prominent efferent innervation that originates in the brainstem and ends as bouton terminals on vestibular hair cells and afferents in each end organ. Although the functional significance of this centrifugal pathway is not well understood, it is clear that efferent neurons, when electrically stimulated under experimental conditions, profoundly impact vestibular afferent discharge. Effects range from chiefly excitation in fish and mammalian vestibular afferents to a more heterogeneous mixture of inhibition and/or excitation in amphibians, reptiles, and birds. What accounts for these diverse response properties? Recent cellular and pharmacological characterization of efferent synaptic mechanisms in turtle offers some insight. In the turtle posterior crista, vestibular efferent neurons are predominantly cholinergic and the effects of efferent stimulation on vestibular afferent discharge can be ascribed to three distinct signaling pathways: (1) Hyperpolarization of type II hair cells mediated by α9/α10-nAChRs and SK-potassium channels; (2) Depolarization of bouton and calyx afferents via α4ß2*-containing nAChRs; and (3) A slow excitation of calyx afferents attributed to muscarinic AChRs. In this review, we discuss the evidence for these pathways in turtle and speculate on their role in mammalian vestibular efferent actions where synaptic mechanisms are largely unknown.

Generation of spinal motor neurons from human fetal brain-derived neural stem cells: role of basic fibroblast growth factor.

Neural stem cells (NSCs) have some specified properties but are generally uncommitted and so can change their fate after exposure to environmental cues. It is unclear to what extent this NSC plasticity can be modulated by extrinsic cues and what are the molecular mechanisms underlying neuronal fate determination. Basic fibroblast growth factor (bFGF) is a well-known mitogen for proliferating NSCs. However, its role in guiding stem cells for neuronal subtype specification is undefined. Here we report that in-vitro-expanded human fetal forebrain-derived NSCs can generate cholinergic neurons with spinal motor neuron properties when treated with bFGF within a specific time window. bFGF induces NSCs to express the motor neuron marker Hb9, which is blocked by specific FGF receptor inhibitors and bFGF neutralizing antibodies. This development of spinal motor neuron properties is independent of selective proliferation or survival and does not require high levels of MAPK activation. Thus our study indicates that bFGF can play an important role in modulating plasticity and neuronal fate of human NSCs and presumably has implications for exploring the full potential of brain NSCs for clinical applications, particularly in spinal motor neuron regeneration.

Compatibility of human fetal neural stem cells with hydrogel biomaterials in vitro.

Stroke and spinal cord or brain injury often result in cavity formation. Stem cell transplantation in combination with tissue engineering has the potential to fill such a cavity and replace lost neurons. Several hydrogels containing unique features particularly suitable for the delicate nervous system were tested by determining whether these materials were compatible with fetal human neural stem cells (hNSCs) in terms of toxicity and ability to support stem cell differentiation in vitro. The hydrogels examined were pluronic F127 (PF127), Matrigel and PuraMatrix. We found that PF127, in a gelated (30%) form, was toxic to hNSCs, and Matrigel, in a gelated (1-50%) form, prevented hNSCs' normal capacity for neuronal differentiation. In contrast, PuraMatrix was the most optimal hydrogel for hNSCs, since it showed low toxicity when gelated (0.25%) and retained several crucial properties of hNSCs, including migration and neuronal differentiation. Further optimization and characterization of PuraMatrix is warranted to explore its full potential in assisting neural regeneration in vivo.

Astrocytes enhance long-term survival of cholinergic neurons differentiated from human fetal neural stem cells.

Establishment of an in vitro model of human cholinergic neurons would be highly desirable for understanding and developing treatment for Alzheimer's and motoneuron diseases. Previously we reported that the combination of basic fibroblast growth factor (bFGF), heparin, and laminin directs human fetal neural stem cells to form cholinergic neurons. One problem, however, is that long-term in vitro survival of these cells is low. Our goal for this study was to determine whether astrocytes or their secreted factors enhance differentiation and survival of cholinergic neurons under long-term differentiation conditions. We demonstrate here that astrocytes or astrocyte conditioned media did not enhance cholinergic differentiation but did increase the long-term survival of differentiated human neural stem cells, particularly cholinergic neurons. We further show that astrocytes protected long-term-differentiated cells from apoptotic cell death, which is at least partially mediated by astrocyte-secreted bFGF. Our findings indicate that long-term survival of human stem cell-derived cholinergic neurons requires trophic factors from nonneuronal cells. This data may provide insights into the development of an in vitro model of long-term cultured human cholinergic neurons useful for understanding of the mechanisms of cholinergic differentiation and developing treatments for neurological diseases.

Identification of early disease progression in an ALS rat model.

Transgenic rat models of amyotrophic lateral sclerosis (ALS) have recently been developed. Most assays of ALS-symptoms in these models monitor disease onset accurately, but do not identify individuals that will develop these symptoms before the motor deficits become apparent. Peak bodyweight has recently been shown to indicate affected individuals before motor deficits become apparent. However, it must be determined retrospectively due to weight fluctuation. Here, we report that exploratory activities detected by a photobeam activity system (PAS) and wire mesh ascending test can be used to detect slight motor deficits in the early phase of ALS. Thus, these tests may be used in addition to peak bodyweight to monitor early disease progression and to assay efficacy of new therapeutic interventions.

Signaling pathways mediating a selective induction of nitric oxide synthase II by tumor necrosis factor alpha in nerve growth factor-responsive cells.

BACKGROUND: Inflammation and oxidative stress play a critical role in neurodegeneration associated with acute and chronic insults of the nervous system. Notably, affected neurons are often responsive to and dependent on trophic factors such as nerve growth factor (NGF). We previously showed in NGF-responsive PC12 cells that tumor necrosis factor alpha (TNFalpha) and NGF synergistically induce the expression of the free-radical producing enzyme inducible nitric oxide synthase (iNOS). We proposed that NGF-responsive neurons might be selectively exposed to iNOS-mediated oxidative damage as a consequence of elevated TNFalpha levels. With the aim of identifying possible therapeutic targets, in the present study we investigated the signaling pathways involved in NGF/TNFalpha-promoted iNOS induction. METHODS: Western blotting, RT-PCR, transcription factor-specific reporter gene systems, mutant cells lacking the low affinity p75NTR NGF receptor and transfections of TNFalpha/NGF chimeric receptors were used to investigate signalling events associated with NGF/TNFalpha-promoted iNOS induction in PC12 cells. RESULTS: Our results show that iNOS expression resulting from NGF/TNFalpha combined treatment can be elicited in PC12 cells. Mutant PC12 cells lacking p75NTR did not respond, suggesting that p75NTR is required to mediate iNOS expression. Furthermore, cells transfected with chimeric TNFalpha/NGF receptors demonstrated that the simultaneous presence of both p75NTR and TrkA signaling is necessary to synergize with TNFalpha to mediate iNOS expression. Lastly, our data show that NGF/TNFalpha-promoted iNOS induction requires activation of the transcription factor nuclear factor kappa B (NF-kappaB). CONCLUSION: Collectively, our in vitro model suggests that cells bearing both the high and low affinity NGF receptors may display increased sensitivity to TNFalpha in terms of iNOS expression and therefore be selectively at risk during acute (e.g. neurotrauma) or chronic (e.g. neurodegenerative diseases) conditions where high levels of pro-inflammatory cytokines in the nervous system occur pathologically. Our results also suggest that modulation of NFkappaB-promoted transcription of selective genes could serve as a potential therapeutic target to prevent neuroinflammation-induced neuronal damage.

Effect of growth factors on proliferation and phenotypic differentiation of human fetal neural stem cells.

Human fetal neural stem cells (hNSCs) can be expanded in vitro by mitogens or growth factors, such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and/or leukemia inhibitory factor (LIF). Their effects on proliferation rate and differentiation pattern of hNSCs, however, have not been fully characterized. In this study, we cultured hNSCs in seven regimens, including bFGF, EGF, and LIF, either alone or in combinations. Cells were maintained as neurospheres in treatment media for various periods, up to six passages. A combination of bFGF, EGF, and LIF expanded hNSCs more efficiently than any other treatment as determined by counting total cell numbers using a trypan blue exclusion assay, a WST-1 cell viability assay, and a bromodeoxyuridine incorporation flow cytometric analysis. Differentiation patterns of hNSCs expanded under different conditions were also analyzed. We reported previously that hNSCs primed in vitro with a combination of bFGF, heparin, and laminin (FHL) induced neuronal differentiation toward a cholinergic phenotype. In this study, we show that the FHL priming increases neuronal differentiation while decreasing astroglial generation in all treatment groups as determined by immunostaining. However, cells proliferated under different growth factor conditions do vary in their phenotypic differentiation patterns. Particularly, significant generation of cholinergic cells was observed only in hNSCs expanded with EGF/bFGF or EGF/bFGF/LIF, but not with other treatment regimens, even when they are exposed to the same priming procedure. Our results indicate that hNSCs are highly plastic, with their proliferation and differentiation potential dependent on different growth factor treatments.