Cryopreservation and functional analysis of cardiac autonomic neurons.

BACKGROUND: Generally, primary neurons are isolated and seeded within hours of isolation, but cryopreservation, documented for a small number of central and peripheral neuronal subtypes, can contribute to improved utility and reduce the cost of developing new in vitro models. The preservation of cells of the autonomic nervous system (ANS), specifically sympathetic and parasympathetic neurons, has not been explored. NEW METHOD: In this work, we establish a method for preserving cardiac ANS neurons as well as evaluating the phenotypical changes of dissociated superior cervical ganglia (sympathetic neurons) and intracardiac ganglia (parasympathetic neurons) for up to a month of storage in liquid nitrogen. RESULTS: Neuron populations maintained a viability of at least 35%, and the extent of neurite outgrowth was not different from fresh cells, regardless of the storage duration studied. Expression of tyrosine hydroxylase and choline acetyl transferase were maintained over one month of cryopreservation in sympathetic and parasympathetic populations, respectively. Electrophysiological recordings for both neuron types indicate sustained characteristic resting potentials, excitability, and action potentials after more than one month in liquid nitrogen. COMPARISON WITH EXISTING METHODS: Primary cultures of the autonomic nervous system have been previously established for in vitro investigations. This is the first example of preserving primary ANS neuron cultures for long-term on-demand use. CONCLUSIONS: This report describes a readily implemented method for cryopreserving sympathetic and parasympathetic neurons that does not alter neither morphological nor electrophysiological characteristics. This methodology expands the utility of ANS cultures for use in morphological and functional assays.

Fibroblast growth factor 23 counters vitamin D metabolism and action in human mesenchymal stem cells.

Chronic kidney disease (CKD) is associated with elevated circulating fibroblast growth factor 23 (FGF23), impaired renal biosynthesis of 1α,25-dihydroxyvitamin D (1α,25(OH)2D), low bone mass, and increased fracture risk. Our previous data with human mesenchymal stem cells (hMSCs) indicated that vitamin D metabolism in hMSCs is regulated as it is in the kidney and promotes osteoblastogenesis in an autocrine/paracrine manner. In this study, we tested the hypothesis that FGF23 inhibits vitamin D metabolism and action in hMSCs. hMSCs were isolated from discarded marrow during hip arthroplasty, including two subjects receiving hemodialysis and a series of 20 subjects (aged 49-83 years) with estimated glomerular filtration rate (eGFR) data. The direct in vitro effects of rhFGF23 on hMSCs were analyzed by RT-PCR, Western immunoblot, and biochemical assays. Ex vivo analyses showed positive correlations for both secreted and membrane-bound αKlotho gene expression in hMSCs with eGFR of the subjects from whom hMSCs were isolated. There was downregulated constitutive expression of αKlotho, but not FGFR1 in hMSCs obtained from two hemodialysis subjects. In vitro, rhFGF23 countered vitamin D-stimulated osteoblast differentiation of hMSCs by reducing the vitamin D receptor, CYP27B1/1α-hydroxylase, biosynthesis of 1α,25(OH)2D3, and signaling through BMP-7. These data demonstrate that dysregulated vitamin D metabolism in hMSCs may contribute to impaired osteoblastogenesis and altered bone and mineral metabolism in CKD subjects due to elevated FGF23. This supports the importance of intracellular vitamin D metabolism in autocrine/paracrine regulation of osteoblast differentiation in hMSCs.

Neural responses to electrical stimulation in 2D and 3D in vitro environments.

Electrical stimulation (ES) to manipulate the central (CNS) and peripheral nervous system (PNS) has been explored for decades, recently gaining momentum as bioelectronic medicine advances. The application of ES in vitro to modulate a variety of cellular functions, including regenerative potential, migration, and stem cell fate, are being explored to aid neural degeneration, dysfunction, and injury. This review describes the materials and approaches for the application of ES to the PNS and CNS microenvironments, towards an improved understanding of how ES can be harnessed for beneficial clinical applications. Emphasized are some recent advances in ES, including conductive polymers, methods of charge transfer, impact on neural cells, and a brief overview of alternative methodologies for cellular targeting including magneto, ultrasonic, and optogenetic stimulation. This review will examine how heterogenous cell populations, including neurons, glia, and neural stem cells respond to a wide range of conductive 2D and 3D substrates, stimulation regimes, known mechanisms of response, and how cellular sources impact the response to ES.