The conventional method for the derivation of human embryonic stem cells (hESCs) involves inner cell mass (ICM) co-culture with a feeder layer of inactivated mouse or human embryonic fibroblasts in an in vitro fertilisation culture dish. Growth factors potentially involved in primary derivation of hESCs may be lost or diluted in such a system. We established a microdrop method which maintained feeder cells and efficiently generated hESCs. Embryos were donated for stem cell research after fully informed patient consent. A feeder cell layer was made by incubating inactivated mouse embryonic fibroblasts (MEFs) feeder cells in a 50 microl drop of medium (DMEM/10% foetal calf serum) under mineral oil in a small tissue culture dish. MEFs formed a confluent layer and medium was replaced with human embryonic stem medium supplemented with 10% Plasmanate (Bayer) and incubated overnight. Cryopreserved embryos were thawed and cultured until the blastocyst stage and the zona pellucida removed with pronase (2 mg/ml; Calbiochem). A zona-free intact blastocyst was placed in the feeder microdrop and monitored for ES derivation with medium changed every 2-3 d. Proliferating hESCs were passaged into other feeder drops and standard feeder preparation by manual dissection until a stable cell line was established. Six hESC lines (Shef 3-8) were derived. From a total of 46 blastocysts (early to expanded), five hESC lines were generated (Shef 3-7). Shef 3-6 were generated on MEFs from 25 blastocysts. Shef7 was generated on human foetal gonadal embryonic fibroblasts from a further 21 blastocysts. From our experience, microdrop technique is more efficient than conventional method for derivation of hESCs and it is much easier to monitor early hESC derivation. The microdrop method lends itself to good manufacturing practice derivation of hESCs.

Thanks to stem cells’ capability to differentiate into multiple cell types, damaged human tissues and organs can be rapidly well-repaired. Therefore, their applicability in the emerging field of regenerative medicine can be further expanded, serving as a promising multifunctional tool for tissue engineering, treatments for various diseases, and other biomedical applications as well. However, the differentiation and survival of the stem cells into specific lineages is crucial to be exclusively controlled. In this frame, growth factors and chemical agents are utilized to stimulate and adjust proliferation and differentiation of the stem cells, although challenges related with degradation, side effects, and high cost should be overcome. Owing to their unique physicochemical and biological properties, graphene-based nanomaterials have been widely used as scaffolds to manipulate stem cell growth and differentiation potential. Herein, we provide the most recent research progress in mesenchymal stem cells (MSCs) growth, differentiation and function utilizing graphene derivatives as extracellular scaffolds. The interaction of graphene derivatives in human and rat MSCs has been also evaluated. Graphene-based nanomaterials are biocompatible, exhibiting a great potential applicability in stem-cell-mediated regenerative medicine as they may promote the behaviour control of the stem cells. Finally, the challenges, prospects and future trends in the field are discussed.

Stem cells provide various potential applications in regenerative medicine through its ability of self-renewal and differentiation. Among the various stem cells, dental pulp stem cells (DPSCs) have shown encouraging results in their ability to regenerate. Honey has been used in traditional culture as a natural medicine in supporting wound healing. Yet, very few studies on honey were conducted for its potential as a proliferative agent for stem cells. The aim of this study is to evaluate the stability of two Trigona spp. honeys (1 and 2) added in culture media and its proliferative effect on DPSCs. Both honeys were diluted with standard culture medium through dilution process to prepare the concentrations of 0.01%, 0.04%, 0.10% and 0.25%. DPSCs were treated with the diluted honeys for 24 hours. The proliferative activity was determined through the images taken using an inverted microscope for every six hours. In addition, the MTT assay was conducted to determine the cell viability of DPSCs when treated with both honey 1 and 2 at various concentrations. The results showed a stable culture media added with honey for three days and a dose-dependent proliferative effect of both Trigona spp. honey samples on DPSCs. Optimum proliferative effects were observed at 24 hours for both Trigona spp. honey 1 and 2 on DPSCs. The optimum concentration of Trigona spp. honey 1 was from 0.04% to 0.10% and Trigona spp. honey 2 was below 0.01%. It is concluded that Trigona spp. honey has a promising proliferative effect on DPSCs.

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Carbon derived from biomass waste usage is rising in various fields of application due to its availability, cost-effectiveness, and sustainability, but it remains limited in tissue engineering applications. Carbon derived from human hair waste was selected to fabricate a carbon-based bioscaffold (CHAK) due to its ease of collection and inexpensive synthesis procedure. The CHAK was fabricated via gelation, rapid freezing, and ethanol immersion and characterised based on their morphology, porosity, Fourier transforms infrared (FTIR), tensile strength, swelling ability, degradability, electrical conductivity, and biocompatibility using Wharton’s jelly-derived mesenchymal stem cells (WJMSCs). The addition of carbon reduced the porosity of the bioscaffold. Via FTIR analysis, the combination of carbon, agar, and KGM was compatible. Among the CHAK, the 3HC bioscaffold displayed the highest tensile strength (62.35 ± 29.12 kPa). The CHAK also showed excellent swelling and water uptake capability. All bioscaffolds demonstrated a slow degradability rate (<50%) after 28 days of incubation, while the electrical conductivity analysis showed that the 3AHC bioscaffold had the highest conductivity compared to other CHAK bioscaffolds. Our findings also showed that the CHAK bioscaffolds were biocompatible with WJMSCs. These findings showed that the CHAK bioscaffolds have potential as bioscaffolds for tissue engineering applications.

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