DJ-1 Can Replace FGF-2 for Long-Term Culture of Human Pluripotent Stem Cells in Defined Media and Feeder-Free Condition.

Conventional human pluripotent stem cell (hPSC) cultures require high concentrations of expensive human fibroblast growth factor 2 (hFGF-2) for hPSC self-renewal and pluripotency in defined media for long-term culture. The thermal instability of the hFGF-2 mandates media change every day, which makes hPSC culture costly and cumbersome. Human DJ-1 (hDJ-1) can bind to and stimulate FGF receptor-1. In this study, for the first time, we have replaced hFGF-2 with hDJ-1 in the essential eight media and maintained the human embryonic stem cells (hESCs), H9, in the defined media at feeder-free condition. After more than ten passages, H9 in both groups still successfully maintained the typical hESC morphology and high protein levels of pluripotency markers, SSEA4, Tra1-60, Oct4, Nanog, and ALP. DNA microarray revealed that more than 97% of the 21,448 tested genes, including the pluripotency markers, Sox2, Nanog, Klf4, Lin28A, Lin28B, and Myc, have similar mRNA levels between the two groups. Karyotyping revealed no chromosome abnormalities in both groups. They also differentiated sufficiently into three germ layers by forming in vitro EBs and in vivo teratomas. There were some variations in the RT-qPCR assay of several pluripotency markers. The proliferation rates and the mitochondria of both groups were also different. Taken together, we conclude that hDJ-1 can replace hFGF-2 in maintaining the self-renewal and the pluripotency of hESCs in feeder-free conditions.

Prokaryotic soluble overexpression and purification of oncostatin M using a fusion approach and genetically engineered E. coli strains.

Human Oncostatin M (OSM), initially discovered as a tumour inhibitory factor secreted from U-937 cells, is a gp130 (IL-6/LIF) cytokine family member that exhibits pleiotropic effects in inflammation, haematopoiesis, skeletal tissue alteration, liver regeneration, cardiovascular and metabolic diseases. Cytoplasmic expression of OSM in Escherichia coli results in inclusion bodies, and complex solubilisation, refolding and purification is required to prepare bioactive protein. Herein, eight N-terminal fusion variants of OSM with hexahistidine (His6) tag and seven solubility-enhancing tags, including thioredoxin (Trx), small ubiquitin-related modifier (Sumo), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilisation substance protein A (Nusa), human protein disulphide isomerase (PDI) and the b'a' domain of PDI (PDIb'a'), were tested for soluble OSM expression in E. coli. The His6-OSM plasmid was also introduced into genetically engineered Origami 2 and SHuffle strains to test expression of the protein. At 18 °C, MBP-tagged OSM was highly expressed and solubility was dramatically enhanced. In addition, His6-OSM was more highly expressed and soluble in Origami 2 and SHuffle strains than in BL21(DE3). MBP-OSM and His6-OSM were purified more than 95% with yields of 11.02 mg and 3.27 mg from a 500 mL culture. Protein identity was confirmed by mass spectroscopy, and bioactivity was demonstrated by in vitro inhibition of Th17 cell differentiation.

A chemical conjugate between HER2-targeting antibody fragment and Pseudomonas exotoxin A fragment demonstrates cytotoxic effects on HER2-expressing breast cancer cells.

Conventionally, immunotoxins have been produced as a single polypeptide from fused genes of an antibody fragment and a toxin. In this study, we adopted a unique approach of chemical conjugation of a toxin protein and an antibody fragment. The two genes were separately expressed in Escherichia coli and purified to high levels of purity. The two purified proteins were conjugated using a chemical linker. The advantage of this approach is its ability to overcome the problem of low recombinant immunotoxin production observed in some immunotoxins. Another advantage is that various combinations of immunotoxins can be prepared with fewer efforts, because the chemical conjugation of components is relatively simpler than the processes involved in cloning, expression, and purification of multiple immunotoxins. As a proof of concept, the scFv of trastuzumab and the PE24 fragment of Pseudomonas exotoxin A were separately produced using E. coli and then chemically crosslinked. The new immunotoxin was tested on four breast cancer cell lines variably expressing HER2. The chemically crosslinked immunotoxin exhibited cytotoxicity in proportion to the expression level of HER2. In conclusion, the present study revealed an alternative method of generating an immunotoxin that could effectively reduce the viability of HER2-expressing breast cancer cells. These results suggest the effectiveness of this method of immunotoxin crosslinking as a suitable alternative for producing immunotoxins. [BMB Reports 2019; 52(8): 496-501].

Nurses' Communication of Safety Events to Nursing Home Residents and Families.

Although communication is an essential part of the nursing process, nurses have little to no formal education in how to best communicate patient safety event (PSE) information to nursing home (NH) residents and their family members. The current mixed-methods study tested an intervention aimed at educating nurses on how to communicate a PSE to residents/family members using a structured communication tool. Nurse participants improved their knowledge of PSE communication, especially about the cause of the event, what they would say to the resident/family member, and future prevention of the PSE. Through qualitative subgroup analysis, an increased number of empathic statements were noted post-intervention. The tool tested in this study provides structure to an important care process that is necessary for improving the culture of safety in NH settings. [Journal of Gerontological Nursing, 44(2), 25-32.].

Generation of functional endothelial-like cells from adult mouse germline-derived pluripotent stem cells.

Functional endothelial cells and their progenitors are required for vascular development, adequate vascular function, vascular repair and for cell-based therapies of ischemic diseases. Currently, cell therapy is limited by the low abundance of patient-derived cells and by the functional impairment of autologous endothelial progenitor cells (EPCs). In the present study, murine germline-derived pluripotent stem (gPS) cells were evaluated as a potential source for functional endothelial-like cells. Cells displaying an endothelial cell-like morphology were obtained from gPS cell-derived embryoid bodies using a combination of fluorescence-activated cell sorting (FACS)-based selection of CD31-positive cells and their subsequent cultivation on OP9 stromal cells in the presence of VEGF-A. Real-time reverse transcriptase polymerase chain reaction, FACS analysis and immunofluorescence staining showed that the gPS cell-derived endothelial-like cells (gPS-ECs) expressed endothelial cell-specific markers including von Willebrand Factor, Tie2, VEGFR2/Flk1, intercellular adhesion molecule 2 and vascular endothelial-cadherin. The high expression of ephrin B2, as compared to Eph B4 and VEGFR3, suggests an arterial rather than a venous or lymphatic differentiation. Their capability to take up Dil-conjugated acetylated low-density lipoprotein and to form capillary-like networks on matrigel confirmed their functionality. We conclude that gPS cells could be a novel source of endothelial cells potentially suitable for regenerative cell-based therapies for ischemic diseases.

Brief report: evaluating the potential of putative pluripotent cells derived from human testis.

Human adult germline stem cells (haGSCs) were established from human testicular biopsies and were claimed to be pluripotent. Recently, the gene expression profile of haGSCs demonstrated that these cells presented with a fibroblast rather than a pluripotent identity. Nevertheless, haGSCs were reported to generate teratomas. In this report, we address this discrepancy. Instead of using haGSCs, which are no longer available for the stem cell community, we used a human testicular fibroblastic cell (hTFC) line that presents with a gene expression profile highly similar to that of haGSCs. Indeed, as shown by microarray analysis, the similarity between hTFCs and haGSCs is comparable to human embryonic stem cell (hESC) lines derived by different laboratories. We argue that the almost identical gene expression profile of hTFCs and haGSCs should result in a very similar if not identical differentiation potential. Strikingly, hTFCs were not able to generate teratomas after injection into nude mice. Instead, they formed a mesenchymal lesion that morphologically resembled the putative haGSC-derived teratomas reported previously. We conclude that haGSCs, which exhibit a profile similar to that of fibroblasts and could not generate teratomas, are not pluripotent. Future work will have to show if pluripotent cells can be derived from human testicular biopsies. Mouse work and certain testicular germ cell tumors indicate that this will be possible.

Human adult germline stem cells in question.

Conrad et al. have generated human adult germline stem cells (haGSCs) from human testicular tissue, which they claim have similar pluripotent properties to human embryonic stem cells (hESCs). Here we investigate the pluripotency of haGSCs by using global gene-expression analysis based on their gene array data and comparing the expression of pluripotency marker genes in haGSCs and hESCs, and in haGSCs and human fibroblast samples derived from different laboratories, including our own. We find that haGSCs and fibroblasts have a similar gene-expression profile, but that haGSCs and hESCs do not. The pluripotency of Conrad and colleagues' haGSCs is therefore called into question.

Conversion of adult mouse unipotent germline stem cells into pluripotent stem cells.

Germline stem cells (GSCs), often called spermatogonial stem cells, are unipotent stem cells that can give rise only to gametes. Under defined culture conditions, unipotent GSCs can be converted into pluripotent stem cells, termed as germline-derived pluripotent stem (gPS) cells. gPS cells can be differentiated into cells forming all three germ layers and germ cells. In this study, we describe a simple and robust protocol for the derivation of GSCs from adult mouse testis and the rapid and reproducible conversion of GSCs into gPS cells. Under our defined culture conditions, GSCs can be converted into gPS cells in approximately 1 month. The initial number of plated GSCs and the culture time are two conditions that are critical for the successful conversion of GSCs into gPS cells. gPS cells are similar to embryonic stem cells, as judged by molecular and cellular properties and its development potential. Thus, generation of gPS cells holds potential for tissue regenerative medicine.