Differential scanning fluorimetry illuminates silk feedstock stability and processability.

The ability to design and implement silk feedstock formulations for tailored spinning has so far eluded the bioengineers. Recently, the high throughput screening technique of differential scanning fluorimetry (DSF) demonstrated the link between the instability transition temperature (Ti) and the processability of the silk feedstock. Using DSF we screened a large set of chemicals known to affect solvent quality. A multivariate analysis of the results shows that, regardless of the diversity of chemicals, three groupings are significantly distinguishable: G1 = similar to native silk; G2 = largely dominated by electrostatic interactions; and G3 = dominated by chelating interactions. We propose a thermodynamic analysis based on a pre- and post-transition fit to estimate the van't Hoff enthalpies (ΔHv) and the instability temperature (Ti). Our analysis shows that the ΔTi and ΔHv values were distinct: G1 (ΔTi = 0.23 ± 0.2; ΔHv = -159.1 ± 5.6 kcal mol(-1)), G2 (ΔTi = -7.3 ± 0.7; ΔHv = -191.4 ± 5.5 kcal mol(-1)), and G3 (ΔTi = -19.9 ± 3.3; ΔHv = -68.8 ± 6.0 kcal mol(-1)). Our analysis further combined the ΔTi value and the ΔHv value using stability ΔΔG to find that G1 only marginally stabilizes native silks (ΔΔG = -0.15 ± 0.04 kcal mol(-1)), whereas G2 and G3 destabilize native silk (ΔΔG = 3.8 ± 0.11 and ΔΔG = 3.8 ± 0.3 kcal mol(-1), respectively). Here our analysis shows that native silk has a complex multistep transition that is possibly non-cooperative. However, all three groupings also show a direct and cooperative transition with varied stabilization effects. This analysis suggests that native silks are able to sample multiple substates prior to undergoing (or to delay) the final transition. We conclude by hypothesizing that the observed energetic plasticity may be mediated by a fragile packaging of the silk tertiary structure that is readily lost when the solvent quality changes

Sacrificial core-based electrospinning: A facile and versatile approach to fabricate devices for potential cell and tissue encapsulation applications

Electrospinning uses an electric field to produce fine fibers of nano and micron scale diameters from polymer solutions. Despite innovation in jet initiation, jet path control and fiber collection, it is common to only fabricate planar and tubular-shaped electrospun products. For applications that encapsulate cells and tissues inside a porous container, it is useful to develop biocompatible hollow core-containing devices. To this end, by introducing a 3D-printed framework containing a sodium chloride pellet (sacrificial core) as the collector and through post-electrospinning dissolution of the sacrificial core, we demonstrate that hollow core containing polyamide 66 (nylon 66) devices can be easily fabricated for use as cell encapsulation systems. ATR-FTIR and TG/DTA studies were used to verify that the bulk properties of the electrospun device were not altered by contact with the salt pellet during fiber collection. Protein diffusion investigations demonstrated that the capsule allowed free diffusion of model biomolecules (insulin, albumin and Ig G). Cell encapsulation studies with model cell types (fibroblasts and lymphocytes) revealed that the capsule supports the viability of encapsulated cells inside the capsule whilst compartmentalizing immune cells outside of the capsule. Taken together, the use of a salt pellet as a sacrificial core within a 3D printed framework to support fiber collection, as well as the ability to easily remove this core using aqueous dissolution, results in a biocompatible device that can be tailored for use in cell and tissue encapsulation applications

Electrosprayed genipin cross‐linked alginate–chitosan microcarriers for ex vivo expansion of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are potential therapeutic candidates, owing to their ability to differentiate into several cell types. However, the gap between availability and demand of MSCs requires alternative expansion methods from 2D culture flasks. Microcarriers are a promising approach for MSC expansion due to their high surface area‐to‐volume ratio. However, current commercial microcarriers do not provide the highest cell yield due to low cell attachment efficiencies and difficulty in cell detachment. This study developed a hydrogel‐based microcarrier from genipin cross‐linked alginate–chitosan beads. Alginate beads were produced by electrospraying before being coated with chitosan and cross‐linked in genipin. The degree of cross‐linking was determined through fluorescence reading of the genipin‐chitosan conjugates. MSCs cultured on these microcarriers had a 26% higher cell attachment and twice the proliferation rate compared to the commercial microcarrier Cytodex 1. Cells easily detached from the hydrogel beads and did not require extended incubation periods or intense agitation during cell harvest. There was no significant difference in gene expression between the two microcarriers for the positive MSC surface markers CD‐90, CD‐105, and CD‐73 as well as showing either low or no signal for negative MSC surface markers CD‐45 and CD‐34

Effect of substrate topography and chemistry on human mesenchymal stem cell markers: a transcriptome study

Background and Objectives:The International Society for Cellular Therapy (ISCT) proposed a set of minimal markers for identifying human mesenchymal stromal cells (hMSCs) in 2007. Since then, with the growing interest of better characterising hMSCs, various additional surface markers have been proposed. However, the impact of how culture conditions, in particular, the culture surface, vary the expression of hMSC markers was overlooked. Methods and Results:In this study, we utilized the RNA sequencing data on hMSCs cultured on different surfaces to investigate the variation of the proposed hMSC biomarkers. One of the three ISCT proposed positive biomarker, CD90 was found to be significantly down regulated on hMSCs culture on fibrous surfaces when compared to flat surfaces. The detected gene expression values for 177 hMSCs biomarkers compiled from the literature are reported here. Correlation and cluster analysis revealed the existence of different biomarker communities that displayed a similar expression profile. We found a list of hMSCs biomarkers which are the least sensitive to a change in surface properties and another list of biomarkers which are found to have high sensitivity to a change in surface properties. Conclusions:This study demonstrated that substrate properties have paramount effect on altering the expressions of hMSCs biomarkers and the proposed list of substrate-stable and substrate-sensitive biomarkers would better assist in the population characterisation. However, proteomic level analysis would be essential to confirm the observations noted

Differential and interactive effects of substrate topography and chemistry on human mesenchymal stem cell gene expression

Variations in substrate chemistry and the micro-structure were shown to have a significant effect on the biology of human mesenchymal stromal cells (hMSCs). This occurs when differences in the surface properties indirectly modulate pathways within numerous signaling networks that control cell fate. To understand how the surface features affect hMSC gene expression, we performed RNA-sequencing analysis of bone marrow-derived hMSCs cultured on tissue culture-treated polystyrene (TCP) and poly(l-lactide) (PLLA) based substrates of differing topography (Fl: flat and Fs: fibrous) and chemistry (Pr: pristine and Am: aminated). Whilst 80% of gene expression remained similar for cells cultured on test substrates, the analysis of differentially expressed genes (DEGs) revealed that surface topography significantly altered gene expression more than surface chemistry. The Fl and Fs topologies introduced opposite directional alternations in gene expression when compared to TCP control. In addition, the effect of chemical treatment interacted with that of topography in a synergistic manner with the Pr samples promoting more DEGs than Am samples in all gene ontology function groups. These findings not only highlight the significance of the culture surface on regulating the overall gene expression profile but also provide novel insights into cell-material interactions that could help further design the next-generation biomaterials to facilitate hMSC applications. At the same time, further studies are required to investigate whether or not the observations noted correlate with subsequent protein expression and functionality of cells

Effect of substrate topography and chemistry on human mesenchymal stem cell markers: a transcriptome study

Background and Objectives:The International Society for Cellular Therapy (ISCT) proposed a set of minimal markers for identifying human mesenchymal stromal cells (hMSCs) in 2007. Since then, with the growing interest of better characterising hMSCs, various additional surface markers have been proposed. However, the impact of how culture conditions, in particular, the culture surface, vary the expression of hMSC markers was overlooked. Methods and Results:In this study, we utilized the RNA sequencing data on hMSCs cultured on different surfaces to investigate the variation of the proposed hMSC biomarkers. One of the three ISCT proposed positive biomarker, CD90 was found to be significantly down regulated on hMSCs culture on fibrous surfaces when compared to flat surfaces. The detected gene expression values for 177 hMSCs biomarkers compiled from the literature are reported here. Correlation and cluster analysis revealed the existence of different biomarker communities that displayed a similar expression profile. We found a list of hMSCs biomarkers which are the least sensitive to a change in surface properties and another list of biomarkers which are found to have high sensitivity to a change in surface properties. Conclusions:This study demonstrated that substrate properties have paramount effect on altering the expressions of hMSCs biomarkers and the proposed list of substrate-stable and substrate-sensitive biomarkers would better assist in the population characterisation. However, proteomic level analysis would be essential to confirm the observations noted

Transcriptomics of human multipotent mesenchymal stromal cells: Retrospective analysis and future prospects

The plastic-adherent, fibroblast-like, clonogenic cells found in the human body now defined as multipotent “Mesenchymal Stromal Cells” (MSCs) hold immense potential for cell-based therapies. Recently, research and basic knowledge of these cells has fast-tracked, both from fundamental and translational perspectives. There have been important discoveries with respect to the available variety of tissue sources, the development of protocols for their easy isolation and in vitro expansion and for directed differentiation into various cell types. In addition, there has been discovery of novel abilities such as immune-modulation and the further development of the use of biomaterials to aid isolation, expansion and differentiation together with improved delivery to the selected optimal tissue site. However, the molecular fingerprint of MSCs in these contexts remains imprecise and inadequate. Consequently, without this crucial knowledge it is difficult to achieve progress to determine with precision their practical developmental potentials. Detailed investigations on the global gene expression, or transcriptome, of MSCs could offer essential clues in this regard. In this article, we address the challenges associated with MSC transcriptome studies, the paradoxes observed in published experimental results and the need for careful transcriptomic analysis. We describe the exemplary applications with various transcriptome platforms that are used to address the variation in biomarkers and the identification of differentiation processes. The evolution and the potentials for adapting next-generation sequencing (NGS) technology in transcriptome analysis are discussed. Lastly, based on review of the existing understanding and published studies, we propose how NGS may be applied to promote further understanding of the biology of MSCs and their use in allied fields such as regenerative medicine

Culture surfaces induce hypoxia-regulated genes in human mesenchymal stromal cells

Culturing human Mesenchymal stromal cells (hMSCs) in vitro in hypoxic conditions resulted in reduced senescence, enhanced pluripotency and altered proliferation rate. It has been known that in vitro hypoxia affects expression of cell surface proteins. However, the impact of culture surfaces on the hypoxia-regulated genes (HRG) have not yet been reported. This study utilized Next-Generation sequencing to analyse the changes in the gene expression levels of HRG for hMSCs cultured on different culture surfaces. The samples, which were cultured on four different synthesized surfaces (treatments) and tissue culture plate (control), resulted in a difference in growth rate. The sequencing results revealed that the transcription of a number of key genes involved in regulating hypoxic functions were significantly altered, including HIF2A, a marker for potency, differentiation, and various cellular functions. Significant alternations in the expression levels of previously reported oxygen-sensitive surface proteins were detected in this study, some of which closely correlate with the expression levels of HIF2A. Our analysis of the hMSCs transcriptome and HRG mapped out a list of genes encoding surface proteins which may directly regulate or be regulated by HIF2A. The findings from this study showed that culture surfaces have an impact on regulating the expression profile of HRG. Therefore, novel culture surfaces may be designed to selectively activate HIF2A and other HRG and pathways under in vitro normoxia. The understanding of the crosstalk between the regulating genes of hypoxia and culture surfaces may be utilized to strengthen desired hypoxic functions