425247 Adult Human Mesenchymal Stem Cell Differentiation at the Population and Single-Cell Levels Under Alternating Electric Current

Monday, November 9, 2015: 5:15 PM
151A/B (Salt Palace Convention Center)
Marissa E. Wechsler1, Brian P. Hermann2 and Rena Bizios1, (1)Department of Biomedical Engineering, The University of Texas at San Antonio, San Antonio, TX, (2)Department of Biology, The University of Texas at San Antonio, San Antonio, TX

Progress in developing clinically-relevant biomedical applications (such as regeneration and repair of damaged bone tissues) using stem cells has been slow because the conditions to generate either the required numbers of undifferentiated cells or lineage-specific cells remain, at best, partially understood. Cells involved in the process of new bone formation include (i) osteoblasts (differentiated, bone-forming cells) and (ii) bone-marrow derived mesenchymal stem cells (undifferentiated, multipotent, with the potential to differentiate at least into osteoblasts, chondrocytes, and adipocytes upon appropriate stimulation).  To date, biochemical compounds (such as bone morphogenetic proteins) have been utilized to promote the differentiation of mesenchymal stem cells into osteoblasts. In contrast, the effects of biophysical stimuli on stem cell functions remain unknown.

For these reasons, the present in vitrostudy used interdisciplinary approaches and novel laboratory setups to examine and optimize the effects of alternating electric current alone (i. e., in the absence of supplemented exogenous growth factors) to promote the  osteodifferentiation of adult human mesenchymal stem cells. Motivation for the study was provided by the physiological milieu: bone and its constituent cells exist, and function, in an environment composed of biochemical as well as by biophysical (specifically, mechanical and electrical) stimuli.

For this purpose, adult human mesenchymal cells were cultured on flat, indium-tin-oxide-coated glass (pre-coated with fibronectin) in the absence of exogenous growth factors. A custom-made laboratory set-up was used to expose adult human mesenchymal stem cells (passage 3-5) under standard cell culture conditions to alternating electric current (sinusoidal waveform, in the ranges of 5-40 μA and 5-10 Hz frequency) for various durations (in the range of 1-24 hours) daily, for up to 21 consecutive days. The cellular/molecular analyses utilized two approaches: (i) at the cell population level; and, for the first time, at the (ii) single-cell level. Quantitative real-time polymerase chain reaction (PCR) was used to determine gene expression in mesenchymal stem cells at the population and single-cell levels. Genes indicative of the lineage specific osteoblastic pathway (specifically, TAZ, RUNX2, SP7, SPP1, and BGLAP), as well of the chondrogenic (COL2A) and adipogenic (FABP4) lineages were monitored.

Optimal osteodifferentiation of adult human mesenchymal stem cells was obtained at the cell population level when cells were exposed to a sinusoidal, 10 μA, 10 Hz, alternating electric current for 6 hours daily for up to 21 consecutive days. Under these conditions the mesenchymal stem cells expressed early (TAZ, RUNX2, and SP7), and late (SPP1 and BGLAP) genes indicative of exclusive osteodifferentiation because genes indicative of the chondrocyte and adipocyte lineages were not expressed at all time points tested. The single-cell analysis provided evidence of the heterogeneity of the adult human mesenchymal stem cells and confirmed expression of genes indicative of the early stages of osteodifferentiation.

In addition to providing fundamental information pertinent to stem cell physiology, the unique biophysical stimulus examined provides a still untapped alternative approach, (which does not require supplemented exogenous growth factors) in order to obtain critically-needed lineage specific differentiated cells (e.g., osteoblasts) for tissue engineering and tissue regeneration applications. In this respect, the present study could have major impact in bioengineering, biotechnology, and in specific therapeutic modalities in the clinical milieu.

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See more of this Session: Stem Cells in Tissue Engineering
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division