Abstract
IntroductionHuman mesenchymal stem cells (hMSCs) have a great clinical potential for tissue regeneration purposes due to its multilineage capability. Previous studies have reported that a single addition of 5-azacytidine (5-AzaC) causes the differentiation of hMSCs towards a myocardial lineage. The aim of this work was to evaluate the effect of 5-AzaC addition frequency on hMSCs priming (i.e., indicating an early genetic differentiation) using two culture environments.MethodshMSCs were supplemented with 5-AzaC while cultured in well plates and in microfluidic chips. The impact of 5-AzaC concentration (10 and 20 μM) and addition frequency (once, daily or continuously), as well as of culture period (2 or 5 days) on the genetic upregulation of PPARγ (adipocytes), PAX3 (myoblasts), SOX9 (chondrocytes) and RUNX2 (osteoblasts) was evaluated.ResultsDaily delivering 5-AzaC caused a higher upregulation of PPARγ, SOX9 and RUNX2 in comparison to a single dose delivery, both under static well plates and dynamic microfluidic cultures. A particularly high gene expression of PPARγ (tenfold-change) could indicate priming of hMSCs towards adipocytes.ConclusionsBoth macro- and microscale cultures provided results with similar trends, where addition frequency of 5-AzaC was a crucial factor to upregulate several genes. Microfluidics technology was proven to be a suitable platform for the continuous delivery of a drug and could be used for screening purposes in tissue engineering research.
Highlights
Human mesenchymal stem cells have a great clinical potential for tissue regeneration purposes due to its multilineage capability
Nowadays there are consolidated in vitro protocols that enable the differentiation of Human mesenchymal stem cells (hMSCs) into mesodermal cell types such as osteoblasts, chondrocytes, adipocytes and myoblasts.[5,6]
The aim of this work was to elucidate the effect of the addition frequency of 5-AzaC on the priming of hMSCs when two culture environments were used, static well plates and dynamic microfluidic cultures
Summary
Human mesenchymal stem cells (hMSCs) have a great clinical potential for tissue regeneration purposes due to its multilineage capability. The impact of 5-AzaC concentration (10 and 20 lM) and addition frequency (once, daily or continuously), as well as of culture period (2 or 5 days) on the genetic upregulation of PPARc (adipocytes), PAX3 (myoblasts), SOX9 (chondrocytes) and RUNX2 (osteoblasts) was evaluated. The multilineage capacity of mesenchymal stem/ stromal cells (MSCs), first suggested by Cohnheim in 1867, resulted in a new paradigm in the field of tissue engineering with high clinical potential.[38] Nowadays there are consolidated in vitro protocols that enable the differentiation of hMSCs into mesodermal cell types such as osteoblasts, chondrocytes, adipocytes and myoblasts.[5,6] In the recent years, the approach of priming hMSCs ( referred as preconditioning or licensing), meaning to prepare the cells for some lineage-specific differentiation, have started to be investigated. One of the main drawbacks of this procedure is that the concentration of the active compound decreases over time, effect linked to its stability and the interaction with cells
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