Cross-striated collagen fibers formation with low amplitude, high frequency vibrations

Human Adipose derived Stem Cells (hASCs) are observed to express genes and proteins associated with the osteoblast phenotype under osteogenic conditions and with mechanical stimulation. New investigations have been carried out to understand the effects of Low Amplitude, High frequency vibrations (HFVs) in vitro. In this work hASCs were cultured in proliferative or osteogenic media and daily unstimulated/stimulated at 30 Hz for 45 minutes for 28 days to evaluate the effects of HFVs on the differentiation of hASCs toward bone tissue. We treated four groups of cells (control in proliferative medium-CP, control in osteogenic medium-CO, treated in proliferative medium-TP and treated in osteogenic medium-TO) for 21 and 28 days. The tissue was incubated in digestion buffer and after suspended in DMEM supplemented with 10% FBS. The hASCs were cultured in proliferative medium (PM) at 37°C with 5% CO2. In order to stimulate the cells, we used a custom made “bioreactor”. At the end of the culture period the DNA content was evaluated, Alizarin red test was used to determine the presence of calcium deposition. Quantitative real-time RT-PCR was performed to check the expression of the investigated osteogenic genes. The extracellular matrix (ECM) extraction to evaluate the amount of the ECM constituents. We quantified the level of the most important osteogenic protein: Col-I, Col-III, osteocalcin (OC), osteopontin (OP), alkaline phopsphatase (ALP), osteonectin (ON) and fibronectin (FN). Ultrastructural investigation with TEM were performed after 21 and 28 days. Statistical analysis: a two-way analysis of variance (ANOVA) was performed. Alizarin red test after 21days culture showed strong differences between treated and control samples. The results of osteogenic gene expression reveal the strong increase of their content (Col-I, OP) for the mechanically treated samples, in both media at 21 days. The results obtained from the protein content analysis at 21 days seem to confirm previous observations: both the osteogenic medium and the mechanical treatment induce the early translation of osteogenic genes. TEM observations showed that in the sample treated with HFVs for 21 days, long collagen fibers of indeterminate length are present; at 28 days fibers appear shorter with the classical arrangement cross-striated in the extracellular space. From our results, the HFV treatment seems to have effect only after 21 days treatment and it seems to be ineffective in the long term trial, only collagen fibers appear more structured after 28 days

Connectivity and circuitry in a dish versus in a brain

In order to understand and find therapeutic strategies for neurological disorders, disease models that recapitulate the connectivity and circuitry of patients’ brain are needed. Owing to many limitations of animal disease models, in vitro neuronal models using patient-derived stem cells are currently being developed. However, prior to employing neurons as a model in a dish, they need to be evaluated for their electrophysiological properties, including both passive and active membrane properties, dynamics of neurotransmitter release, and capacity to undergo synaptic plasticity. In this review, we survey recent attempts to study these issues in human induced pluripotent stem cell-derived neurons. Although progress has been made, there are still many hurdles to overcome before human induced pluripotent stem cell-derived neurons can fully recapitulate all of the above physiological properties of adult mature neurons. Moreover, proper integration of neurons into pre-existing circuitry still needs to be achieved. Nevertheless, in vitro neuronal stem cell-derived models hold great promise for clinical application in neurological diseases in the future

A Time Course Analysis of the Electrophysiological Properties of Neurons Differentiated from Human Induced Pluripotent Stem Cells (iPSCs)

Many protocols have been designed to differentiate human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) into neurons. Despite the relevance of electrophysiological properties for proper neuronal function, little is known about the evolution over time of important neuronal electrophysiological parameters in iPSC-derived neurons. Yet, understanding the development of basic electrophysiological characteristics of iPSC-derived neurons is critical for evaluating their usefulness in basic and translational research. Therefore, we analyzed the basic electrophysiological parameters of forebrain neurons differentiated from human iPSCs, from day 31 to day 55 after the initiation of neuronal differentiation. We assayed the developmental progression of various properties, including resting membrane potential, action potential, sodium and potassium channel currents, somatic calcium transients and synaptic activity. During the maturation of iPSC-derived neurons, the resting membrane potential became more negative, the expression of voltage-gated sodium channels increased, the membrane became capable of generating action potentials following adequate depolarization and, at day 48–55, 50% of the cells were capable of firing action potentials in response to a prolonged depolarizing current step, of which 30% produced multiple action potentials. The percentage of cells exhibiting miniature excitatory post-synaptic currents increased over time with a significant increase in their frequency and amplitude. These changes were associated with an increase of Ca2+ transient frequency. Co-culturing iPSC-derived neurons with mouse glial cells enhanced the development of electrophysiological parameters as compared to pure iPSC-derived neuronal cultures. This study demonstrates the importance of properly evaluating the electrophysiological status of the newly generated neurons when using stem cell technology, as electrophysiological properties of iPSC-derived neurons mature over time

Connectivity and circuitry in a dish versus in a brain

In order to understand and find therapeutic strategies for neurological disorders, disease models that recapitulate the connectivity and circuitry of patients’ brain are needed. Owing to many limitations of animal disease models, in vitro neuronal models using patient-derived stem cells are currently being developed. However, prior to employing neurons as a model in a dish, they need to be evaluated for their electrophysiological properties, including both passive and active membrane properties, dynamics of neurotransmitter release, and capacity to undergo synaptic plasticity. In this review, we survey recent attempts to study these issues in human induced pluripotent stem cell-derived neurons. Although progress has been made, there are still many hurdles to overcome before human induced pluripotent stem cell-derived neurons can fully recapitulate all of the above physiological properties of adult mature neurons. Moreover, proper integration of neurons into pre-existing circuitry still needs to be achieved. Nevertheless, in vitro neuronal stem cell-derived models hold great promise for clinical application in neurological diseases in the future

Spontaneous calcium transients increase in iPSC-derived neurons during development.

<p>A) After imaging of spontaneous Ca²⁺- transients iPSC-derived neurons were fixed and stained for Tuj1 at day 32 and day 55, revealing more complex morphology at day 55 (scale bar = 40 µm). B) Representative Fluo-4 spontaneous Ca²⁺- transients from 3 iPSC-derived neurons at day 32 and at day 55 (ΔFluo = ΔFluorescence; a.u. = arbitrary unit). Neurons exhibit slow broad transients, consistent with early developing neurons at day 32. By day 55, calcium transients take on a more spike-like morphology reflecting what has been reported for maturing neurons. Group data demonstrates that iPSC-derived neurons exhibit a significant increase in calcium transients at day 55 as compared to day 32 (n = 113 neurons/10 coverslips for both groups, p<0.001 comparing day 32 with day 55). C) The average number of Ca²⁺ transients at day 55 was dramatically reduced following perfusion with 1 µM TTX with recovery following washout of the toxin (n = 20/3 coverslips, p<0.001 compared with average number of Ca²⁺ transients prior to TTX perfusion). A sample trace derived from a representative experiment is shown on the left).</p

Evolution of synaptic activity due to AP-independent release of neurotransmitters over time in iPSC-derived neurons plated on POL.

<p>A) Example of continuous recording from an iPSC-derived neuron at 55 days (holding potential at −70 mV). The inset shows an enlarged view of two spontaneous post-synaptic events. B) The number of cells showing synaptic events over the total number of cells recorded in the same day. Cells were recorded at −70 mV in 1 µM TTX in voltage clamp. C) Increase over time of the frequency of the mEPSCs, measured as number of spontaneous events/minute. D) Evolution of the mEPSC amplitude with a voltage clamp at −70 mV from t1 to t3.</p

iPSCs-derived neurons plated with glia express different electrophysiological properties than neurons plated on POL.

<p>A) Representative APs in response to step current injections of 20 pA in current clamp mode for a cell at day 55 co-cultured with mouse glia. APs were observed on 13 out of 15 cells, and in 4 out of the 13 cells with APs rebound APs at the end of hyperpolarizing current injections were also present. B) Examples of mEPSCs recorded in voltage clamp configuration. Cells were plated on mouse glia by mixing them before plating. They were held at −70 mV. TTX at a concentration of 1 µM was added to the bath solution to suppress spontaneous excitation and to allow isolation of synaptic events induced by spontaneous transmitter release.</p

Timeline of the experiments.

<p>The different steps of the experiments are represented, as well as the time-points for the electrophysiological, calcium imaging, immunofluorescence and FACS analyses. The 3 different timing groups for statistical purposes are the following: t1  =  from day 31 to day 38; t2  =  from day 41 to day 45; t3  =  from day 48 to day 55.</p

Evolution of basal membrane properties over time in iPSC-derived neurons plated on POL.

<p>A) The average resting membrane potential (RMP in mV) decreased over time. The number of cells examined for these experiments are shown inside the bar in these and the following figures. The vertical lines correspond to the SEM in this and the following figures. **: p<0.001; *: 0.0010.05 for these and the following figures. B) The membrane input resistance decreased over time (GΩ). C) The membrane time constant decreased over time (ms).</p