Fahsai Kantawong

Nanotopographical Control of Stem Cell Differentiation

Stem cells have the capacity to differentiate into various lineages, and the ability to reliably direct stem cell fate determination would have tremendous potential for basic research and clinical therapy. Nanotopography provides a useful tool for guiding differentiation, as the features are more durable than surface chemistry and can be modified in size and shape to suit the desired application. In this paper, nanotopography is examined as a means to guide differentiation, and its application is described in the context of different subsets of stem cells, with a particular focus on skeletal (mesenchymal) stem cells. To address the mechanistic basis underlying the topographical effects on stem cells, the likely contributions of indirect (biochemical signal-mediated) and direct (force-mediated) mechanotransduction are discussed. Data from proteomic research is also outlined in relation to topography-mediated fate determination, as this approach provides insight into the global molecular changes at the level of the functional effectors.

Proteomic analysis of human osteoprogenitor response to disordered nanotopography

Previous studies have shown that microgroove-initiated contact guidance can induce bone formation in osteoprogenitor cells (OPGs) and produce changes in the cell proteome. For proteomic analysis, differential in-gel electrophoresis (DIGE) can be used as a powerful diagnostic method to provide comparable data between the proteomic profiles of cells cultured in different conditions. This study focuses on the response of OPGs to a novel nanoscale pit topography with osteoinductive properties compared with planar controls. Disordered near-square nanopits with 120 nm diameter and 100 nm depth with an average 300 nm centre-to-centre spacing (300 nm spaced pits in square pattern, but with ±50 nm disorder) were fabricated on 1×1 cm2 polycaprolactone sheets. Human OPGs were seeded onto the test materials. DIGE analysis revealed changes in the expression of a number of distinct proteins, including upregulation of actin isoforms, beta-galectin1, vimentin and procollagen-proline, 2-oxoglutarate 4-dioxygenase and prolyl 4-hydroxylase. Downregulation of enolase, caldesmon, zyxin, GRASP55, Hsp70 (BiP/GRP78), RNH1, cathepsin D and Hsp27 was also observed. The differences in cell morphology and mineralization are also reported using histochemical techniques.

Differential in-gel electrophoresis (DIGE) analysis of human bone marrow osteoprogenitor cell contact guidance

We have used a recent comparative proteomics technique, differential in-gel electrophoresis (DIGE), to study osteoprogenitor cell response to contact guidance in grooves. In order to increase protein output from small sample sizes, we used bioreactor culture before protein extraction and gel electrophoresis. Mass spectroscopy was used for protein identification. A number of distinct proteins were observed to exhibit significant changes in expression. These changes in protein expression suggest that the cells respond to tailored grooved topographies, with alterations in their proteome concurrent with changes in osteoprogenitor phenotype.

Effects of a surface topography composite with puerariae radix on human STRO-1-positive stem cells

Human skeletal stem cells (STRO-1 positive/STRO-1+) respond to different topographical features in various ways. On a flat surface these cells spread and tend to develop a fibroblast-like morphology. On a microgrooved surface enriched skeletal stem cell populations prefer to stretch along the grooves, which affects their cellular structure and differentiation, a phenomenon known as contact guidance. Growth factors, hormones and chemicals can also stimulate cell differentiation. A traditional Chinese medicine, puerariae radix, has previously been observed to stimulate bone formation. The active ingredients have been identified as isoflavones with estrogen-like bioactivity. This study combined the effects of microgrooved topology and hormone-like isoflavones in the biodegradable polymer polycaprolactone (PCL). Human osteogenic cells (STRO-1+) were cultured on flat PCL, grooved PCL and puerariae powder-impregnated grooved PCL for 5 weeks. Coomassie staining indicated that cell growth and survival was similar on flat PCL, grooved PCL and grooved PCL impregnated with 1 wt.% or 2 wt.% puerariae powder. Grooved PCL impregnated with 2 wt.% puerariae powder was observed to have an influence on protein expression, as observed by positive osteocalcin staining. Protein expression profiles were analyzed by difference gel electrophoresis to identify proteins that showed modulation of expression in response to these different environments. Overall, our results suggest that puerariae powder has an additive effect, along with microgrooved topographical stimulation, to promote changes in the STRO-1+ proteome that affect cell phenotype.

Preventing and troubleshooting artefacts in saturation labelled fluorescence 2‐D difference gel electrophoresis (saturation DiGE)

Saturation DiGE is a powerful but challenging technique for the characterisation of changes in protein expression between two or more scarce samples. In this paper, measures to prevent and troubleshoot artefacts in the saturation DiGE workflow are discussed, with illustration of some examples as they may be encountered in gel images or analysis.

Protein Expression of STRO-1 Cells in Response to Different Topographic Features

Human skeletal stem cells (STRO-1 positive) display distinct responses to different topographical features. On a flat surface, skeletal cells spread, and in vitro, they typically display a polarized, fibroblast-like morphology. However, on microgrooved surfaces, these cells prefer to stretch along the grooves forming a similar morphology to in vivo, bipolarized fibroblasts. In contrast, on nanopits, these cells display a polygonal and osteoblastic phenotype. We have examined mechanotransduction events of STRO-1 positive in response to fibroblastic, microgrooved and osteogenic, controlled disorder nanopit, topographies using proteomics after 3 days in culture. Protein expression profiles were analyzed by difference gel electrophoresis to identify proteins that showed modulation of expression in response to different topographic features to assess early decision events in these cells on these discrete topographies. After only 72 hours in culture, STRO-1 positive displayed differential regulations of families of proteins involved in cell migration and proliferation. The current study indicated that osteogenic decision specific events had already occurred. Runx2 was localized in nuclei of the skeletal stem cells on the osteogenic nanopits; however, few signaling pathway changes were observed. This study demonstrated that micro- and nanotopographies activated skeletal stem cells at different times and with distinct mechanotransduction profiles.

Characterization of Gelatin Composite with Low Content Hydroxyapatite and the Influence on Mesenchymal Stem Cell Culture

In tussue engineering, hydrogel-based scaffold is one of the most common method for bone tissue engineering. Gelatin is a common material for scaffold, whereas hydroxyapatite (HA) has a similar composition and structure to natural bone mineral. HA can also increase cell adhesion ability of the scaffold. This research focuses on the fabrication of hydrogel scaffolds using gelatin composite with nanocrystalline hydroxyapatite (nHA). Then the mechanical and physical caharacteristics of the scaffold is investigetad. Low contents nHA is introduced into gelatin in order to modulate mesenchymal stem cell (MSC) behavior. There are three types of scaffolds which contain various HA content. The gelatin is crosslinked with glutaraldehyde before freeze-drying. The Young’s modulus of the surface is investigated using Atomic force microscopy (AFM). The pore size is investigated using scanning electron microscope (SEM). Human MSCs are culture on the scaffold for 3 weeks. The result shows the sucesse in cell cultivation. However, the human MSCs cultured on the fabricated hydrogels do not show any lineage-specific differentiation.

Variation of Hydroxyapatite Content in Soft Gelatin Affects Mesenchymal Stem Cell Differentiation

Gelatin is a common material used in tissue engineering and hydroxyapatite (HA) has a composition and structure similar to natural bone mineral. HA is also used to increase the adhesion ability of scaffolds. The physical and mechanical properties of gelatin, together with the chemical properties of HA, can affect cell differentiation. The main purpose of this study is to investigate the gene expression of human mesenchymal stem cells (HMSCs) upon culturing on gelatin composite with HA. Low amounts of HA were introduced into the gelatin in order to modulate properties of gelatin. Three types of hydrogel were fabricated by glutaraldehyde crosslinking before lyophilization to produce the porous 3D structure: (1) pure gelatin, (2) 0.5 mg/ml HA in gelatin, and (3) 1 mg/ml HA in gelatin. The fabricated hydrogels were used as scaffolds to cultivate HMSCs for two periods - 24 hours and 3 weeks. The results showed that all types of fabricated hydrogels could be used to cultivate HMSCs. Changes of gene expressions indicated that the HMSCs cultured on the 1 mg/ml HA in gelatin showed neuronal lineage-specific differentiation.

Reprogramming of mouse fibroblasts into neural lineage cells using biomaterials.

Introduction: Induced neural stem cells (iNSCs) have the ability of differentiation into neurons, astrocytes and oligodendrocytes. iNSCs are very useful in terms of research and treatment. The present study offers an idea that biomaterials could be one of the tools that could modulate reprogramming process in the fibroblasts. Methods: Gelatin biomaterials were fabricated into 3 types, including (i) gelatin, (ii) gelatin with 1 mg/mL hydroxyapatite, and (iii) gelatin with hydroxyapatite and pig brain. NIH/3T3 fibroblasts were cultured on each type of biomaterial for 7, 9 and 14 days. RT-PCR was performed to investigate the gene expression of the fibroblasts on biomaterials compared to the fibroblasts on tissue culture plates. PI3K/Akt signaling was performed by flow cytometry after 24 hours seeding on the biomaterials. The biomaterials were also tested with the human APCs and PDL cells. Results: The fibroblasts exhibited changes in the expression of the reprogramming factor; Klf‫4 and the neural transcription factors; NFIa, NFIb and Ptbp1 after 9 days culture. The cultivation of fibroblasts on the biomaterials for 7 days showed a higher expression of the transcription factor SOX9. The expression of epigenetic genes; Kat2a and HDAC3 were changed upon the cultivation on the biomaterials for 9 days. The fibroblasts cultured on the biomaterials showed an activation of PI3K/Akt signaling. The human APCs and human PDL cells developed mineralization process on biomaterials Conclusion: Changes in the expression of Klf4, NFIa, NFIb, Ptbp1 and SOX9 indicated that fibroblasts were differentiated into an astrocytic lineage. It is possible that the well-designed biomaterials could work as powerful tools in the reprogramming process of fibroblasts into iNSCs.