Functional Equivalency in Human Somatic Cell Nuclear Transfer-Derived Endothelial Cells.

The derivation of human embryonic stem cells (hESCs) by somatic cell nuclear transfer (SCNT) has prompted a re-emerging interest in using such cells for therapeutic cloning. Despite recent advancements in derivation protocols, the functional potential of CHA-NT4 derived cells is yet to be elucidated. For this reason, this study sought to differentiate CHA-NT4 cells toward an endothelial lineage in order to evaluate in vitro and in vivo functionality. To initial differentiation, embryoid body formation of CHA-NT4 was mediated by concave microwell system which was optimized for hESC-endothelial cell (EC) differentiation. The isolated CD31+ cells exhibited hallmark endothelial characteristics in terms of morphology, tubule formation, and ac-LDL uptake. Furthermore, CHA-NT4-derived EC (human nuclear transfer [hNT]-ESC-EC) transplantation in hind limb ischemic mice rescued the hind limb and restored blood perfusion. These findings suggest that hNT-ESC-EC are functionally equivalent to hESC-ECs, warranting further study of CHA-NT4 derivatives in comparison to other well established pluripotent stem cell lines. This revival of human SCNT-ESC research may lead to interesting insights into cellular behavior in relation to donor profile, mitochondrial DNA, and oocyte quality. Stem Cells 2019;37:623-630.

Examination of endothelial cell-induced epidermal regeneration in a mice-based chimney wound model.

As wound contraction in the cutaneous layer occurs rapidly in mice, mechanical means are typically used to deliberately expose the wound to properly investigate healing by secondary intention. Previously, silicon rings and splinting models were attempted to analyze histological recovery but prevention of surrounding epidermal cell migration and subsequent closure was minimal. Here, we developed an ideal chimney wound model to evaluate epidermal regeneration in murine under hESC-EC transplantation through histological analysis encompassing the three phases of regeneration: migration, proliferation, and remodeling. Human embryonic stem cell derived endothelial cells (hESC-EC) were transplanted due to possessing a well-known therapeutic effect in angiogenesis which also enhances epidermal repair to depict the process of regeneration. Following a standard 1 mm biopsy punch, a chimney manufactured by modifying a 1.7 mL microtube was simply inserted into the excisional wound to complete the modeling process. Under this model, the excisional wound remained fully exposed for 14 days and even after 4 weeks, only a thin transparent layer of epidermal tissue covered the wound site. This approach is able to more accurately depict epidermal repair in relation to histology while also being a user-friendly and cost-effective way to mimic human recovery in rodents and evaluate epithelial repair induced by a form of therapy.

Well-defined differentiation of hESC-derived hemangioblasts by embryoid body formation without enzymatic treatment.

Human hemangioblasts exist only during the early embryonic developmental stage thereby limiting the adult cellular source from which to obtain such cells for study. To overcome this, hemangioblast studies have focused on utilizing human embryonic stem cell (hESC) derivatives but current methods are cell-line dependent. Single cell dissociation of a hESC colony quickly led to cell death in most hESC lines due to enzyme treatment which, in turn, reduced induction potential and hemangioblast differentiation efficiency. Therefore, we sought to effectively improve the process of cell dissociation that is adaptable to various hESC lines and increase the initial induction potential of embryoid body (hEB). As a result, we determined an effective cell dissociation method through a comparison study involving various reagents which demonstrated successful dissociation regardless of cell line and enhanced hemangioblast differentiation efficiency.

Modification to the injection needle to a screw needle improves effective cell delivery in acute myocardial infarction.

Evaluation of therapeutic effects of transplanted cells in ischemic heart failure models are important issues. However, traditional injection needles that are widely used in clinical practice tend to reduce the amount of functional cells relative to the injected amount. We now describe a cell transplantation technique using a screw needle. After inducing acute myocardial infarction in a rat model, human embryonic stem cell-derived endothelial cells were injected into the infarcted regions with a screw or straight-curved needle. When an equal volume of cells was transplanted, the screw group suffered minimal cell loss, showed improvement in LV wall thickness (74.5 ± 6.2 vs. 64.4 ± 7.8 %), epicardium scar length (19.3 ± 2.8 vs. 24.6 ± 6.4 %), and area of engraft. Thus, even a simple change in the structure of an instrument can have a large impact on transplantation efficiency.

Effective method for the isolation and proliferation of primary lung cancer cells from patient lung tissues.

We have developed a technique for isolating and culturing primary lung cancer cells extracted from patient tissue to facilitate anti-cancer drug development. Patient-derived lung cancer tissues were mechanically dissociated to 40-100 µm. Dispase was then used to isolate cultured lung cancer cell populations, which were re-plated on Matrigel-coated dishes containing N2-supplemented medium and growth factors. This method allows pure populations of primary non-small cell lung cancer cells to be grown in vitro. The isolated cells exhibited hallmark cancerous properties such as abnormal chromosomes and in vivo tumor formation. The cell lines generated through this procedure may help to advance our knowledge of certain forms of lung cancer and may also be useful for developing patient-specific anti-cancer drug screening procedures.

Sneezing and asymptomatic virus transmission.

The novel coronavirus disease (COVID-19) spread pattern continues to show that geographical barriers alone cannot contain a virus. Asymptomatic carriers play a critical role in the nature of this virus quickly escalating into a global pandemic. Asymptomatic carriers may transmit the virus unintentionally through sporadic sneezing. A novel Computational Fluid Dynamics (CFD) approach has been proposed with a realistic modeling of a human sneeze achieved by the combination of state-of-the-art experimental and numerical methods. This modeling approach may be suitable for future engineering analyses aimed at reshaping public spaces and common areas, with the main objective to accurately predict the spread of aerosol and droplets that may contain pathogens. This study shows that the biomechanics of a human sneeze, including complex muscle contractions and relaxations, can be accurately modeled by the angular head motion and the dynamic pressure response during sneezing. These have been considered as the human factors and were implemented in the CFD simulation by imposing a momentum source term to the coupled Eulerian-Lagrangian momentum equations. The momentum source was modeled by the measured dynamic pressure response in conjunction with the angular head motion. This approach eliminated the need to create an ad hoc set of inlet boundary conditions. With this proposed technique, it is easier to add multiple fixed and/or moving sources of sneezes in complex computational domains. Additionally, extensive sensitivity analyses based on different environmental conditions were performed, and their impact was described in terms of potential virus spread.

Cellular organization of three germ layer cells on different types of noncovalent functionalized graphene substrates.

Graphene and its derivatives have seen a rapid rise in interest as promising biomaterials especially in the field of tissue engineering, regenerative medicine, and cell biology of late. Despite its proven potential in numerous biological applications, information regarding the relationship between the different forms of graphene and cell lineages is still lacking partly due to its topical emergence in cellular studies. Herein, we explore the biocompatibility of four types of graphene substrates (chemical vapor deposition grown graphene, mechanically exfoliated graphene, chemically exfoliated graphene oxide, and reduced graphene oxide) with three types of somatic cells (keratinocytes, hepatocytes, endothelial cells) derived from the three germ layers in relation to cell adhesion, proliferation, morphology, and gene expression. The results revealed exceptional cell adhesion for all tested groups but enhanced proliferation and cytoskeletal interconnectivity in graphene oxide and reduced graphene oxide substrates. We were unable to detect any adverse effects in gene expression and survivability during a week of culture. We further show topographic changes to graphene substrates under fetal bovine serum adsorption to better illustrate the actual microenvironment of inhabitant cells. This study highlights the extraordinary synergy between graphene and somatic cells, suggesting the discretionary use of extracellular matrix components for in vitro cultivation.

Mechanotransduction of human pluripotent stem cells cultivated on tunable cell-derived extracellular matrix.

Cell-derived matrices (CDM) are becoming an attractive alternative to conventional biological scaffolding platforms due to its unique ability to closely recapitulate a native extracellular matrix (ECM) de novo. Although cell-substrate interactions are recognized to be principal in regulating stem cell behavior, very few studies have documented the acclimation of human pluripotent stem cells (hPSCs) on pristine and altered cell-derived matrices. Here, we investigate crosslink-induced mechanotransduction of hPSCs cultivated on decellularized fibroblast-derived matrices (FDM) to explore cell adhesion, growth, migration, and pluripotency in various biological landscapes. The results showed either substrate-mediated induction or inhibition of the Epithelial-Mesenchymal-Transition (EMT) program, strongly suggesting that FDM stiffness can be a dominant factor in mediating hPSC plasticity. We further propose an optimal FDM substratum intended for long-term hPSC cultivation in a feeder-free niche-like microenvironment. This study carries significant implications for hPSC cultivation and encourages more in-depth studies towards the fundamentals of hPSC-CDM interactions.

Effect of BMP-2 Delivery Mode on Osteogenic Differentiation of Stem Cells.

Differentiation of stem cells is an important strategy for regeneration of defective tissue in stem cell therapy. Bone morphogenetic protein-2 (BMP-2) is a well-known osteogenic differentiation factor that stimulates stem cell signaling pathways by activating transmembrane type I and type II receptors. However, BMPs have a very short half-life and may rapidly lose their bioactivity. Thus, a BMP delivery system is required to take advantage of an osteoinductive effect for osteogenic differentiation. Previously, BMP delivery has been designed and evaluated for osteogenic differentiation, focusing on carriers and sustained release system for delivery of BMPs. The effect of the delivery mode in cell culture plate on osteogenic differentiation potential was not evaluated. Herein, to investigate the effect of delivery mode on osteogenic differentiation of BM-MSCs in this study, we fabricated bottom-up release and top-down release systems for culture plate delivery of BMP-2. And also, we selected Arg-Gly-Asp- (RGD-) conjugated alginate hydrogel for BMP-2 delivery because alginate is able to release BMP-2 in a sustained manner and it is a biocompatible material. After 7 days of culture, the bottom-up release system in culture plate significantly stimulated alkaline phosphate activity of human bone marrow-mesenchymal stem cells. The present study highlights the potential value of the tool in stem cell therapy.

Directing human embryonic stem cells towards functional endothelial cells easily and without purification.

Hemangioblasts or blood islands only arise in early development thereby the sources to obtain these bi-potential cells are limited. While previous studies have isolated both lineages in vitro through the hemangioblast, derivation efficiency was rather low due to cellular damage attributed by enzyme usage and fluorescent activated cell sorting (FACS). This study focused on avoiding the use of damaging factors in the derivation of endothelial cells (ECs). Single cell H9-human embryonic stem cells (hESCs) were obtained by using a mild dissociation protocol then human embryoid body (hEB) formation was performed under hemangioblast differentiation conditions. The hEBs were subjected to a two-stage cytokine treatment procedure. Subsequent culture of the adhesive cells in day 4 hEBs gave arise to a seemingly pure population of ECs. The hESC-derived ECs were characterized by identifying signature endothelial gene and protein markers as well as testing for in vitro functionality. Furthermore, in vivo functionality was also confirmed by transplanting the cells in hindlimb ischemic murine models. We demonstrate that the genetic change required for EC derivation precedes blast colony formation. Furthermore, cell damage was prevented by abating enzyme usage and FACS, resulting in a high yield of ECs upon adhesion. Under this method, confluent cultures of ECs were obtainable 4 days after hEB formation which is significantly faster than previous protocols.

Pertussis toxin enhances colony organization of enzymatic-dissociated single human embryonic stem cells.

Human embryonic stem cells (hESCs) self-renew indefinitely as highly organized pluripotent colonies. Unlike mouse pluripotent stem cell colonies, human colonies form a uniform, flat, epithelium-like monolayer. Interestingly, it has been reported that colony morphology is closely correlated with the maintenance of pluripotency. However, the molecular mechanisms that underlie human pluripotent colony formation and organization are poorly understood. In this study, we used real-time imaging tools to examine the in vitro colony formation of enzymatically dissociated single hESCs under feeder-free conditions. We demonstrate that colony formation consists of 4 stages: attachment, migration, aggregation, and colony formation, which are facilitated in an intracellular, calcium-dependent manner. Moreover, we found that blocking G(i)-coupled G protein-coupled receptor (GPCR) signaling results in enhanced cell-cell interactions and plays an integral role in promoting the survival of hESCs in culture. From the imaging results, we identified the conditions required for colony formation, and we identified the importance of blocking G(i)-coupled GPCR by pertussis toxin in modulating hESC colony formation and organization. These results will likely be useful for engineering hESCs to further study the mechanisms involved in their function.

A comparison of human cord blood- and embryonic stem cell-derived endothelial progenitor cells in the treatment of chronic wounds.

Endothelial progenitor cells (EPCs) promote new blood vessel formation and increase angiogenesis by secreting growth factors and cytokines in ischemic tissues. Therefore, EPCs have been highlighted as an alternative cell source for wound healing. EPCs can be isolated from various sources, including the bone marrow, cord blood, and adipose tissue. However, several recent studies have reported that isolating EPCs from these sources has limitations, such as the isolation of insufficient cell numbers and the difficulty of expanding these cells in culture. Thus, human embryonic stem cells (hESCs) have generated great interest as an alternative source of EPCs. Previously, we established an efficient preparation method to obtain EPCs from hESCs (hESC-EPCs). These hESC-EPCs secreted growth factors and cytokines, which are known to be important in angiogenesis and wound healing. In this study, we directly compared the capacity of hESC-EPCs and human cord blood-derived EPCs (hCB-EPCs) to benefit wound healing. The number of hESC-EPCs increased during culture and was always higher than the number of hCB-EPCs during the culture period. In addition, the levels of VEGF and Ang-1 secreted by hESC-EPCs were significantly higher than those produced by hCB-EPCs. After transplantation in a mouse dermal excisional wound model, all EPC-transplanted wounds exhibited better regeneration than in the control group. More importantly, we found that the wounds transplanted with hESC-EPCs showed significantly accelerated re-epithelialization. Thus, hESC-EPCs may be a promising cell source for the treatment of chronic wounds.