Multipotent progenitor cells from the adult human brain: neurophysiological differentiation to mature neurons

Abstract

It was long held as an axiom that new neurons are not produced in the adult human brain. More recent studies have identified multipotent cells whose progeny express glial or neuronal markers. This discovery may lead to new therapeutic strategies for CNS disorders, either by stimulating neurogenesis in vivo or by transplanting multipotent progenitor cells (MPCs) that have been propagated and differentiated in vitro. The clinical application of such approaches will be limited by the ability of these cells to develop into functional neurons. To facilitate an understanding of mechanisms regulating neurogenesis in the adult human brain, we characterized the developmental processes MPCs go through when progressing to a neuron. Human tissue was harvested during temporal lobe resections because of epilepsy, and cells were cultured as neurospheres. Our findings demonstrate that at an early stage, these cells often stain with neuronal markers without possessing any functional neuronal properties. Over a period of 4 weeks in culture, cells go through characteristic steps of morphological and electrophysiological development towards functional neurons; they develop a polarized appearance with multiple dendrites, whereas the membrane potential becomes more negative and the input resistance decreases [from -48 +/- 10 mV/557 +/- 85 MOmega (n = 15) between days 7 and 11 to -59 +/- 9 mV/380 +/- 79 MOmega (n = 9) between days 25 and 38, respectively]. Active membrane properties were first observed on day 7 and consisted of a voltage-gated K+-current. Later in the second week the cells developed voltage-gated Ca2+-channels and fired small Ca2+-driven action potentials. Immature Na+-driven action potentials developed from the beginning of the third week, and by the end of the fourth week the cells fired repetitive action potentials with a completely mature waveform generated by the combined action of the voltage-gated ionic channels INa, IA and IK. After 4 weeks, the newly formed neurons also communicated by the use of GABAergic and glutamatergic synapses. The adult human brain thus harbours MPCs, which have the ability to develop into neurons and in doing this follow characteristic steps of neurogenesis as seen in the developing brain.

Membrane Properties of Neuron-Like Cells Generated from Adult Human Bone-Marrow-Derived Mesenchymal Stem Cells

“…This variation indicated that the neuron-like cells had a wide range of different sizes and=or electrical characteristics that could reflect differences in their developmental stages or the types of neurons being generated. The passive membrane properties of the neuron-like cells were similar to previously published data for neurons differentiated from human and rodent embryonic stem cells [27,29], human and rodent NSCs [28,[30][31][32], and rodent MeSCs [33]. Although the passive properties revealed that the NSC-like cells might have differentiated into a diverse population of neuron-like cells, it did not indicate anything about their functional characteristics.…”

“…A majority of the action potentials that we detected had small overshoots and brief afterhyperpolarizations. The shape of the action potentials recorded from the neuron-like cells resembled the immature action potentials that are commonly observed for developing human neurons [26] and neuron-like cells differentiating from stem cells isolated from both humans [14,25,28,30] or from rodents [29,32,33]. In addition, the patch clamp recordings were atypical for mature neurons in that the spikes were not blocked by the sodium channel inhibitor TTX at concentrations up to 10 mM, and we did not observe any repetitive spikes or spontaneous action potentials.…”

“…Normally, the sodium current is much smaller than the potassium current in astrocytes and they rarely fire action potentials [45,46]. Besides, similar immature action potentials and current-voltage relationships are often observed for developing human neurons [26] and neuron-like cells differentiated from stem cells isolated from humans [14,25,28,30,37,47,48] or from rodents [29,32,33,49,50]. Finally, consistent with those previously published studies [9,[11][12][13][14]25,47,48,[51][52][53][54], we confirmed that neuron-like cells with neuronal morphology usually express neuronal and not glial markers (Fig.…”

“…This is similar to the time course for the differentiation of both multipotent progenitor cells isolated from adults and embryonic stem cells. Multipotent progenitor cells isolated from human or mouse brains [30,32] or mouse embryonic stem cells [50] do not usually express mature action potentials until they are cultured >2 weeks. This gradual development is consistent with the immunocytochemical analysis of neuronal differentiation markers under similar culture conditions.…”

Derivation of neural precursor cells from human ES cells at 3% O2 is efficient, enhances survival and presents no barrier to regional specification and functional differentiation

“…The frequency of these action potentials depended on the amount of current injected (Figure 3c), as expected for functional neurons. 38 There was also evidence of spontaneous activity in neurons after 30 days of differentiation at 3% O 2 ( Figure 3e). These findings were remarkably similar to the electrophysiological maturation of neurons observed at 20% O 2 (Supplementary Figure 2), clearly demonstrating that there was no maturation block at low oxygen.…”