The conventional method for producing embryonic stem (ES) cell-derived knockout or transgenic mice involves injection of ES cells into normal, diploid blastocysts followed by several rounds of breeding of resultant chimeras and thus is a timeconsuming and inefficient procedure. F0 ES cell pups can also be derived directly from tetraploid embryo complementation, which requires fusion of two-cell embryos. Recently, F0 ES cell pups have been produced by injection of ES cells into eight-cell embryos using a laser-assisted micromanipulation system. We report a simple method for producing F0 ES cell germlinecompetent mice by piezo injection of ES cells into four-or eight-cell embryos. The efficiency of producing live, transgenic mice by this method is higher than that with the tetraploid blastocyst complementation method. This efficient and economical technique for directly producing F0 ES cell offspring can be applicable in many laboratories for creating genetically manipulated mice using ES cell technology and also for stringent testing of the developmental potency of new ES cell or other types of pluripotent stem cell lines.

Mammalian parthenogenetic embryos are not viable and die because of defects in placental development and genomic imprinting. Parthenogenetic ESCs (pESCs) derived from parthenogenetic embryos might advance regenerative medicine by avoiding immuno-rejection. However, previous reports suggest that pESCs may fail to differentiate and contribute to some organs in chimeras, including muscle and pancreas, and it remains unclear whether pESCs themselves can form all tissue types in the body. We found that derivation of pESCs is more efficient than of ESCs derived from fertilized embryos, in association with reduced mitogen-activated protein kinase signaling in parthenogenetic embryos and their inner cell mass outgrowth. Furthermore, in vitro culture modifies the expression of imprinted genes in pESCs, and these cells, being functionally indistinguishable from fertilized embryo-derived ESCs, can contribute to all organs in chimeras. Even more surprisingly, our study shows that live parthenote pups were produced from pESCs through tetraploid embryo complementation, which contributes to placenta development. This is the first demonstration that pESCs are capable of full-term development and can differentiate into all cell types and functional organs in the body.

Authentic bovine embryonic stem (ES) cell lines have not been established despite progress made for more than two decades. Isolation and culture of primary ES cell colonies are the first critical step towards establishment of stable ES cell lines. Here we report a novel method designated as "Separate and Seed" that contributes remarkably to efficient derivation of bovine primary ES-like cell colonies from blastocysts. These primary cultured bovine ES-like cells exhibit morphology typical of ES cells and express pluripotent molecular markers including Oct4, Nanog and alkaline phosphatase. Interestingly, bovine primary ES-like cell colonies distinctively express both stage-specific embryonic antigens 1 and 4 (SSEA1 and SSEA4), unlike mouse and human ES cells. These pluripotent markers may be used for characterization of authentic bovine ES cell lines in later studies. In contrast, whole embryos or inner cell mass (ICM) used for primary culture by conventional methods fails to produce primary bovine ES cell colonies that express all pluripotent stem cell markers shown above. Furthermore, bFGF improves growth and maintained undifferentiated state of bovine ES-like cells for several passages, whereas LIF and ERK inhibitor PD98059 known to promote pluripotency of mouse ES cells are unable to sustain bovine ES-like cells. Although continued efforts are required for improving long-term culture of bovine ES cells, this novel "Separate and Seed" method provides an initial effective step that may eventually lead to derivation of authentic bovine ES cell lines.

Mammalian parthenogenetic embryos (pE) are not viable due to placental deficiency, presumably resulting from lack of paternally expressed imprinted genes. Pluripotent parthenogenetic embryonic stem (pES) cells derived from pE could advance regenerative medicine by avoiding immuno-rejection and ethical roadblocks. We attempted to explore the epigenetic status of imprinted genes in the generation of pES cells from parthenogenetic blastocysts, and its relationship to pluripotency of pES cells. Pluripotency was evaluated for developmental and differentiation potential in vivo, based on contributions of pES cells to chimeras and development to day 9.5 of pES fetuses complemented by tetraploid embryos (TEC). Consistently, pE and fetuses failed to express paternally expressed imprinted genes, but pES cells expressed those genes in a pattern resembling that of fertilized embryos (fE) and fertilized embryonic stem (fES) cells derived from fE. Like fE and fES cells, but unlike pE or fetuses, pES cells and pES cell-fetuses complemented by TEC exhibited balanced methylation of Snrpn, Peg1 and U2af1-rs1. Coincidently, global methylation increased in pE but decreased in pES cells, further suggesting dramatic epigenetic reprogramming occurred during isolation and culture of pES cells. Moreover, we identified decreased methylation of Igf2r, Snrpn, and especially U2af1-rs1, in association with increased contributions of pES cells to chimeras. Our data show that in vitro culture changes epigenetic status of imprinted genes during isolation of pES cells from their progenitor embryos and that increased expression of U2af1-rs1 and Snrpn and decreased expression of Igf2r correlate with pluripotency of pES cells.

Objective The aim of this study was to assess the efficacy of canine umbilical cord mesenchymal stem cells (UC-MSCs) on the treatment of knee osteoarthritis in dogs. Methods Eight dogs were evenly assigned to two groups. The canine model of knee osteoarthritis was established by surgical manipulation of knee articular cartilage on these eight dogs. UC-MSCs were isolated from umbilical cord Wharton's jelly by 0.1% type collagenase I and identified by immunofluorescence staining and adipogenic and osteogenic differentiation in vitro. A suspension of allogeneic UC-MSCs (1 × 106) and an equal amount of physiological saline was injected into the cavitas articularis in the treated and untreated control groups, respectively, on days 1 and 3 posttreatment. The structure of the canine knee joint was observed by magnetic resonance imaging (MRI), B-mode ultrasonography, and X-ray imaging at the 3rd, 7th, 14th, and 28th days after treatment. Concurrently, the levels of IL-6, IL-7, and TNF-α in the blood of the examined dogs were measured. Moreover, the recovery of cartilage and patella surface in the treated group and untreated group was compared using a scanning electron microscope (SEM) after a 35-day treatment. Results Results revealed that the isolated cells were UC-MSCs, because they were positive for CD44 and negative for CD34 surface markers, and the cells were differentiated into adipocytes and osteoblasts. Imaging technology showed that as treatment time increased, the high signal in the MRI T2-weighted images decreased, the echo-free space in B ultrasonography images disappeared basically, and the continuous linear hypoechoic region at the trochlear sulcus thickened. On X-ray images, the serrate defect at the ventral cortex of the patella improved, and the low-density gap of the ventral patella and trochlear crest gradually increased in the treated group. On the contrary, the high signal in the MRI T2-weighted images and the echo-free space in B ultrasonography images still increased after a 14-day treatment in the untreated control group, and the linear hypoechoic region was discontinuous. On the X-ray images, there was no improvement in the serrate defect of the ventral cortex of the patella. Results for inflammatory factors showed that the blood levels of IL-6, IL-7, and TNF-α of the untreated control group were significantly higher than those of the treated group (P < 0.05) 7–14 days posttreatment. The result of SEM showed that the cartilage neogenesis in the treated group had visible neonatal tissue and more irregular arrangement of new tissue fibers than that of the untreated control group. Furthermore, more vacuoles but without collagen fibers were observed in the cartilage of the untreated control group, and the thickness of the neogenetic cartilage in the treated group (65.13 ± 5.29, 65.30 ± 5.83) and the untreated control group (34.27 ± 5.42) showed a significant difference (P < 0.01). Conclusion Significantly higher improvement in cartilage neogenesis and recovery was observed in the treated group compar...

Mesenchymal stem cells (MSCs) are the most promising seed cells for cell therapy. Comparing the biological and transcriptome gene characteristics of MSCs from different sources provides an important basis for the screening of clinically used cells. The main purpose of this experiment was to establish methods for the isolation and culture of MSCs from five different canine sources, including adipose tissue, bone marrow, umbilical cord, amniotic membrane, and placenta, and compare biological and transcriptome characteristics of MSCs, in order to provide a basis for the clinical application of canine MSCs. MSCs were isolated from Chinese pastoral dogs, and the following experiments were performed: (1) the third, sixth, and ninth generations of cells were counted, respectively, and a growth curve was plotted to calculate the MSC population doubling time; (2) the expression of CD34 and CD44 surface markers was studied by immunofluorescence; (3) the third generation of cells were used for osteogenetic and adipogenic differentiation experiments; and (4) MSC transcriptome profiles were performed using RNA sequencing. All of the five types of MSCs showed fibroblast-like adherent growth. The cell surface expressed CD44 instead of CD34; the third-generation MSCs had the highest proliferative activity. The average population doubling time of adipose mesenchymal stem cells (AD-MSCs), placenta mesenchymal stem cells (P-MSCs), bone marrow mesenchymal stem cells (BM-MSCs), umbilical cord mesenchymal stem cells (UC-MSCs), and amniotic mesenchymal stem cells (AM-MSCs) were 15.8 h, 21.2 h, 26.2 h, 35 h, and 41.9 h, respectively. All five types of MSCs could be induced to differentiate into adipocytes and osteoblasts in vitro, with lipid droplets appearing after 8 days and bone formation occurring 5 days after AD-MSC induction. However, the multilineage differentiation for the remaining of MSCs was longer compared to that of the AD-MSCs. The MSC transcriptome profiles showed that AD-MSC and BM-MSCs had the highest homology, while P-MSCs were significantly different compared to the other four types of MSCs. All the isolated MSCs had the main biological characteristics of MSCs. AD-MSCs had the shortest time for proliferation, adipogenesis, and osteogenic differentiation.

The pandemic has changed the way people work, and more and more people are choosing to work from home (WFH). Unlike traditional work patterns, this approach has limitations and has had a significant impact on both organizations and individuals. It also brings many challenges to the work of HR practitioners. HR practitioners, as key players in strategic human resource management, need to take advantage of management innovations under the crisis to improve employees’ work flexibility and effectively address the impact of working from home. This study aims to address the need for employee skill improvement, psychological stress relief, work-family balance, and company culture reinforcement from an HRM perspective because of the impact of WFH work patterns during the COVID-19 crisis.

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