Functional analysis of neuron-like cells differentiated from neural stem cells derived from bone marrow stroma cells in vitro

Abstract

The transversal differentiation of bone marrow stroma cell (BMSCs) into neural stem cells (NSCs) has attracted much attention in recent years because of their therapeutic potential. However, the problem in therapeutic application of NSCs was how to confirm whether neuron-like cells differentiated from bone marrow stroma cell-derived neural stem cells (BMSCs-D-NSCs) possess corresponding functions of neurochemistry and electrophysiology. In the present study, we tried to affirm the function of neuron-like cells differentiated from BMSCs-D-NSCs in vitro. The BMSCs were harvested by gradient centrifugation in Ficoll-Paque and cultured in "NSCs medium". Immunocytochemistry was used to detect positive expression of neuron-specific nuclear protein (NeuN) in neuron-like cells derived from the BMSCs-D-NSCs. High-pressure liquid chromatography (HPLC) was used to identify neuron-like cells by detecting excitable amino acids [aspartic acid (Asp), glutamic acid (Glu)], inhibited amino acids [glycine (Gly), gamma (gamma) -aminobutyric acid (GABA), alanine (Ala)] or monoamines [noradrenaline (NE), 5-hydroxytryptamine (5-HT), dopamine (DA)]. Electrophysiological properties of the neuron-like cells were also examined using patch clamp analysis to verify their neuron-like functions. It was found that the neuron-like cells differentiated from the BMSCs-D-NSCs could express positive NeuN, synthesize and excrete amino acids, and show some typical electrophysiological properties including the typical Na+ and K+ ion channel membrane current under the voltage patch clamp condition, the typical static electrical membrane potential under the current patch clamp condition, and the differential membrane capacitance and resistance values in series between undifferentiated BMSCs-D-NSCs and differentiated neuron-like cells under the whole-cell patch clamp condition. The neuron-like cells differentiated from BMSCs-D-NSCs exhibit both neuron-like biochemical function and some corresponding electrophysiological properties.

Fig. 1

Differentiation of rabbit (a, b) and rhesus monkey (c, d) BMSCs-D-NSCs. (a) The differentiated neuron-like cells (blue arrows) derived from rabbit BMSCs-D-NSCs at Day 20 following culture purification in NSCs medium with retinoic acid (RA, 0.5μg/ml), showed positive NeuN expression. (400×) (b) Typical neuron-like cell (blue arrow) derived from rabbit BMSCs-D-NSCs stained positive for NeuN at Day 22 after being cultured in NSCs medium with RA (0.5μg/ml) and glial cell line-derived neurotrophic factor (GDNF, 20 ng/ml). (400×) (c) The differentiated neuron-like cells derived from rhesus monkey BMSCs-D-NSCs at Day 18 after purifying culture in NSCs medium. The neurite of neuron-like cells connects with each other. (200×) (d) Neuron-like cells derived from rhesus monkey BMSCs-D-NSCs at Day 22 after being cultured in NSCs medium. (400×)

Fig. 2

Chromatograms of Asp, Glu, Gly, Ala and GABA during BMSCs-D-NSCs development and differentiation

Fig. 3

Chromatograms of NE-energy neuron-like cells (NLCs) and controls during BMSCs-D-NSCs development and differentiation

Fig. 4

Analysis of whole current on neuron-like cell membrane derived from rhesus BMSCs-D-NSCs. Under the voltage clamp mode, joint current on cellular membranes was recorded (with ACSF as the extracellular water, and electrode solution as the one testing membrane properties) with no K⁺ current block. An inward current, which was voltage dependent on both fast activation and fast deactivation, and a sequential strong outward current were recorded at −90 mv of command potential, within 20 ms time course of amplified repolarization from −70 mv to + 50 mv

Fig. 5

Analysis of Na⁺ current on neuron-like cell membrane derived from rhesus BMSCs-D-NSCs. Under the voltage clamp mode, an inward current that was voltage dependent on both fast activation and fast deactivation was induced and then recorded in parts of neuron-like cells (8/24), which was reversibly intercepted by 0.5 μm of TTX. It implied that the recorded current was a Na⁺ current, which was activated at the repolarization time from −50 mv to −40 mv and arrived peak value at −20–0 mv, with the average peak value of Na⁺ current 1098.2 ± 182.4 pA. No inward current was detected in the undifferentiated cells (18)

Fig. 6

Analysis of K⁺ current on rhesus BMSCs NSCs and neuron-like cell membrane. Under the voltage clamp mode, parts of undifferentiated cells (8/13) showed K⁺ current with outward rectification (A), but the intensity of the current peak value (712.50 ± 64.08 pA, eight cases) was less than the one in the differentiated neuron-like cells (1059.38± 97.36pA, 16cases) (B). There was a significant difference between the undifferentiated and differentiated cells (P < 0.01). On the base of recording outward current, which was voltage dependent under clamp voltage mode, primitive current (C) was recorded as the delayed rectification K⁺ current after adding 5 mmol/l 4-AP into the extracellular fluid in order to obstruct the fast activated K⁺ current. As soon as 30 mM TEA was added to block the delayed rectification of K⁺ current, the primitive current (D) was recorded as the fast activated transient outward K⁺ current. It implied the existence of K⁺ current passageways in the neuron-like cells