Nanopore-Based Direct RNA-Sequencing Reveals a High-Resolution Transcriptional Landscape of Porcine Reproductive and Respiratory Syndrome Virus

The TRS-mediated discontinuous transcription process is a hallmark of Arteriviruses. Precise assessment of the intricate subgenomic RNA (sg mRNA) populations is required to understand the kinetics of viral transcription. It is difficult to reconstruct and comprehensively quantify splicing events using short-read sequencing, making the identification of transcription-regulatory sequences (TRS) particularly problematic. Here, we applied long-read direct RNA sequencing to characterize the recombined RNA molecules produced in porcine alveolar macrophages during early passage infection of porcine reproductive and respiratory syndrome virus (PRRSV). Based on sequencing two PRRSV isolates, namely XM-2020 and GD, we revealed a high-resolution and diverse transcriptional landscape in PRRSV. The data revealed intriguing differences in subgenomic recombination types between the two PRRSVs while also demonstrating TRS-independent heterogeneous subpopulation not previously observed in Arteriviruses. We find that TRS usage is a regulated process and share the common preferred TRS in both strains. This study also identified a substantial number of TRS-mediated transcript variants, including alternative-sg mRNAs encoding the same annotated ORF, as well as putative sg mRNAs encoded nested internal ORFs, implying that the genetic information encoded in PRRSV may be more intensively expressed. Epigenetic modifications have emerged as an essential regulatory layer in gene expression. Here, we gained a deeper understanding of m5C modification in poly(A) RNA, elucidating a potential link between methylation and transcriptional regulation. Collectively, our findings provided meaningful insights for redefining the transcriptome complexity of PRRSV. This will assist in filling the research gaps and developing strategies for better control of the PRRS.

Establishment and Functional Characterization of Immortalized Rabbit Dermal Papilla Cell Lines

Background Hair follicle (HF) undergo periodic growth and development in mammals, which regulated by dermal papilla cells (DPCs) are reported to play an important role in the HF morphogenesis and development. However, primary DPCs have low proliferative activity, age quickly, and fresh cell isolation is both time-consuming and laborious. Method In this study, we introduced SV40LT into dissociated early passage rabbit vibrissae DPCs with lentiviral vectors and established seven immortalized DP cell lines (R-1, R-2, R-3, R-4, R-5, R-6 and R-7). Result These cell lines displayed early passage morphology and displayed high alkaline phosphatase activity. RT-PCR and immunofluorescence staining showed that all the immortalized cell lines expressed the DPC markers (α-SMA ,IGF1, ALPL, FGF2, BMP2 and TGFβ2; α-SMA and VIM protein), but α-SMA was only expressed well in R-3, R-4, and R-7. Furthermore, it was found that R-7 was the only line to survive beyond 50 passages. Compared to melanoma cells, R-7 did not undergo malignant transformation. Karyotyping and cell growth viability analysis illustrated that the R-7 cell line preserved the basic characteristics of primary DPCs. Conclusion The R-7 DPCs established have potential application for future hair research. The study provides the theoretical basis in the cell research of HF growth and development.

Epigenetic integrity of paternal imprints enhances the developmental potential of androgenetic haploid embryonic stem cells

The use of two inhibitors of Mek1/2 and Gsk3β (2i) promotes the generation of mouse diploid and haploid embryonic stem cells (ESCs) from the inner cell mass of biparental and uniparental blastocysts, respectively. However, a system enabling long-term maintenance of imprints in ESCs has proven challenging. Here, we report that the use of a two-step a2i (alternative two inhibitors of Src and Gsk3β, TSa2i) derivation/culture protocol results in the establishment of androgenetic haploid ESCs (AG-haESCs) with stable DNA methylation at paternal DMRs (differentially DNA methylated regions) up to passage 60 that can efficiently support generating mice upon oocyte injection. We also show coexistence of H3K9me3 marks and ZFP57 bindings with intact DMR methylations. Furthermore, we demonstrate that TSa2i-treated AG-haESCs are a heterogeneous cell population regarding paternal DMR methylation. Strikingly, AG-haESCs with late passages display increased paternal-DMR methylations and improved developmental potential compared to early-passage cells, in part through the enhanced proliferation of H19-DMR hypermethylated cells. Together, we establish AG-haESCs that can long-term maintain paternal imprints.

Red blood cells and their releasates compromise bone marrow-derived human mesenchymal stem/stromal cell survival in vitro

Purpose The use of bone marrow aspirate (BMA) and bone marrow aspirate concentrate (BMC) in the treatment of inflammatory orthopedic conditions has become a common practice. The therapeutic effect of BMA/BMC is thought to revolve primarily around the mesenchymal stem/stromal cell (MSC) population residing within the nucleated cell fraction. MSCs have the unique ability to respond to site of injury via the secretion of immunomodulating factors, resolving inflammation in diseased joints. Recently, the importance of hematocrit (HCT) in BMC has been debated, as the potential impact on MSC function is unknown. In the present study, we investigate MSC health over a short time-course following exposure to a range of HCT and red blood cell releasate (RBCrel) conditions. Methods Bone marrow-derived human MSCs in early passage were grown under conditions of 0%, 2.5%, 5%, 10%, 20% and 40% HCT and RBCrel conditions for 3 days. At each day, the percentage of viable, apoptotic and necrotic MSCs was determined via flow cytometry. Relative viable MSC counts in each condition was determined to account for dynamic changes in overall MSC densities over the time-course. Statistical analysis was performed using a one-way ANOVA comparing test conditions to the control followed by a Dunnett’s multiple comparison test. Results Significant reductions in viable MSCs concurrent with an increase in necrotic MSCs in high HCT and RBCrel conditions was observed within 24 h. At each successive timepoint, the percent and relative number of viable MSCs were reduced, becoming significant in multiple HCT and RBCrel conditions by Day 3. Necrosis appears to be the initial mode of MSC death following exposure to HCT and RBCrel, followed by apoptosis in surviving MSC fractions. Conclusion Various levels of HCT and RBCrel severely compromise MSC health within 3 days and HCT should be controlled in the preparation of BMC products. Further, HCT of BMCs should be routinely recorded and tracked with patient outcomes along with routine metrics (e.g. nucleated cell counts, fibroblast-colony forming units). Differences in HCT may account for the inconsistencies in the efficacy of BMC reported when treating orthopedic conditions.

The Immunoregulatory Effect of Mechanical Stress on Three-dimensional Cultured Mesenchymal Stem Cells After Extensive Expansion

Background: Mesenchymal stem cells (MSCs) have been used to treat immunopathy, and three-dimensional (3D) cultured MSCs show enhanced immunomodulatory property compared with those in two-dimensional (2D) culture. However, both the regulatory mechanisms remain unclear. The aim of the study was to investigate the role of mechanical stress in maintaining the immunomodulatory function of 2D and 3D cultured MSCs.Methods: Umbilical cord mesenchymal stem cells (UC-MSCs) were plated on tissue culture plastic (TCP) as 2D culture and 3D cultured UC-MSCs were seeded in matrigel. Surface markers, clonogenicity, proliferation and immunoregulatory property of UC-MSCs were evaluated. Meanwhile, we established the mouse models of colitis and type 1 diabetes mellitus (T1DM) to reveal the pharmacotherapeutic effects of 3D cultured MSCs in vivo. The effect of changing mechanical stress by modulating Yes-associated protein (YAP) on immunomodulatory function of 2D and 3D cultured UC-MSCs was evaluated by immunofluorescent analysis, real-time quantitative polymerase chain reaction (qPCR) and western blot.Results: We verified early passage UC-MSCs in 2D and 3D cultures exhibited stemness, immunomodulatory property and therapeutic efficacy against immunopathy. However, these characteristics of 2D cultured UC-MSCs were impaired after extensive expansion, whereas 3D culture extended them for several passages by activating YAP. Moreover, prostaglandin E2 (PGE2) could up-regulate YAP to improve the immunomodulatory ability of 2D cultured UC-MSCs after extensive expansion. Conclusions: This work found for the first time that the significance of mechanical stress in maintaining immunoregulatory function of 2D and 3D cultured UC-MSCs, providing a new idea for improving the efficacy of MSCs-based immunotherapy.

Changes in Methylation Patterns of Tumor Suppressor Genes during Extended Human Embryonic Stem Cell Cultures

While studies on embryonic stem cells have been actively conducted, little is known about the epigenetic mechanisms in human embryonic stem cells (hESCs) in extended culture systems. Here, we investigated whether CpG island (CGI) methylation patterns of 24 tumor suppressor genes could be maintained during extended hESC cultures. In total, 10 hESC lines were analyzed. For each cell line, genomic DNA was extracted from early and late passages of cell cultures. CGI methylation levels of 24 tumor suppressor genes were analyzed using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), pyrosequencing, and real-time polymerase chain reaction (PCR). Different CGI methylation patterns of CASP8, FHIT, and CHFR genes were identified in between early and late passages in some hESC lines. CGI methylation levels of CASP8 significantly increased at late passage in CHA-36, CHA-40, and CHA-42 cell lines compared to those at early passage. The CGI methylation of the FHIT gene was higher at late passage than at early passage in CHA-15, CHA-31, CHA-32, and iPS (FS)-1 cell lines but decreased at the late passage in CHA-20 and H1 cell lines. Different CGI methylation patterns were detected for the CHFR gene only in iPS (FS)-1, and the level significantly increased at late passage. Thus, our findings show that CGI methylation patterns could be altered during prolonged ESC cultures and examining these epigenetic changes is important to assess the maintenance, differentiation, and clinical usage of stem cells.

Preliminary Evaluation of Proliferation, Wound Healing Properties, Osteogenic and Chondrogenic Potential of Dental Pulp Stem Cells Obtained from Healthy and Periodontitis Affected Teeth

Background: Dental pulp tissue within the central cavity of the tooth is composed of dental pulp stem cells (DPSC). These mesenchymal stem cells have good proliferative as well as differentiation potential. DPSC has been isolated even from teeth with inflamed pulps and is found to retain their proliferative and differentiation potential. Little research is available about the viability and differentiation potential of DPSC obtained from teeth with periodontitis. In the present study, the aim was to compare the morphological features, stem cell marker (MSC) expression, proliferation rate, migratory and wound healing properties, osteogenic and chondrogenic differentiation potential of DPSCs obtained from periodontally healthy teeth (hDPSCs) and periodontitis affected teeth (pDPSCs). Methods: Dental pulp tissue was obtained from periodontally healthy volunteers (n = 3) and patients with periodontitis undergoing extraction of mobile teeth (n = 3). DPSC were isolated using the explant technique and cultured. All the experiments were performed at early passage (Passage 2), late passage (Passage 6) and after cryopreservation. Morphological features of the hDPSCs and pDPSCs were ascertained using microscopy. The expression of cell surface stem cell markers was assessed by the flow cytometry method. The proliferation and growth rate of the cells were assayed by plotting a growth curve from 0–13 days of culture. The migratory characteristics were assessed by wound scratch assay. Osteogenic and chondrogenic differentiation of the cells was assessed using standard protocols with and without induction. Results: DPSCs were successfully obtained from periodontally healthy teeth (hDPSC) and periodontitis-affected teeth (pDPSCs). The data suggests that there were no morphological differences observed in early passage cells between the two cohorts. Cryopreservation did change the morphology of pDSPCs. There was no significant difference in the positive expression of mesenchymal markers CD73, CD90 and CD105 in early passage cells. However, serial passaging and cryopreservation affected the marker expression in pDPSCs. A faint expression of hematopoietic stem cell markers CD34, CD45 and MHC class II antigen HLA-DR was observed in both the cell types. The expression of HLA-DR is upregulated in pDPSCs compared to hDPSC. A significantly slower growth rate and slower wound healing properties was observed in pDPSCs compared to hDPSC. In late passage and after cryopreservation, the migratory ability of pDPSCs was found to be increased drastically. There was no significant difference in osteogenic potential between the two cell types. However, the chondrogenic potential of pDPSCs was significantly lower compared to hDPSc. Yet, pDPSCs showed enhanced osteogenesis and chondrogenesis at late passage as well as after cryopreservation. Conclusion: The results of this novel study shed light on the isolation of viable DPSC from periodontitis-affected teeth. These cells exhibit a slower growth rate and migratory characteristics compared to their healthy counterparts. There was no difference in osteogenic potential but a reduction in chondrogenic potential was seen in pDPSCs compared to hDPSC. The findings reveal that DPSC from periodontitis-affected teeth presents an easy and viable option for regenerative medicine application. Some additional nutritive factors and protocols may be required to attain better regenerative benefits while using pDPSCs.

Cancer Cells Can Exhibit a Sparing FLASH Effect at Low Doses Under Normoxic In Vitro-Conditions

BackgroundIrradiation with ultra-high dose rate (FLASH) has been shown to spare normal tissue without hampering tumor control in several in vivo studies. Few cell lines have been investigated in vitro, and previous results are inconsistent. Assuming that oxygen depletion accounts for the FLASH sparing effect, no sparing should appear for cells irradiated with low doses in normoxia.MethodsSeven cancer cell lines (MDA-MB-231, MCF7, WiDr, LU-HNSCC4, HeLa [early passage and subclone]) and normal lung fibroblasts (MRC-5) were irradiated with doses ranging from 0 to 12 Gy using FLASH (≥800 Gy/s) or conventional dose rates (CONV, 14 Gy/min), with a 10 MeV electron beam from a clinical linear accelerator. Surviving fraction (SF) was determined with clonogenic assays. Three cell lines were further studied for radiation-induced DNA-damage foci using a 53BP1-marker and for cell cycle synchronization after irradiation.ResultsA tendency of increased survival following FLASH compared with CONV was suggested for all cell lines, with significant differences for 4/7 cell lines. The magnitude of the FLASH-sparing expressed as a dose-modifying factor at SF=0.1 was around 1.1 for 6/7 cell lines and around 1.3 for the HeLasubclone. Similar cell cycle distributions and 53BP1-foci numbers were found comparing FLASH to CONV.ConclusionWe have found a FLASH effect appearing at low doses under normoxic conditions for several cell lines in vitro. The magnitude of the FLASH effect differed between the cell lines, suggesting inherited biological susceptibilities for FLASH irradiation.

Diverse mechanisms activate the PI 3-kinase/mTOR pathway in melanomas: implications for the use of PI 3-kinase inhibitors to overcome resistance to inhibitors of BRAF and MEK

Background The PI 3-kinase (PI3K) pathway has been implicated as a target for melanoma therapy. Methods Given the high degree of genetic heterogeneity in melanoma, we sought to understand the breadth of variation in PI3K signalling in the large NZM panel of early passage cell lines developed from metastatic melanomas. Results We find the vast majority of lines show upregulation of this pathway, and this upregulation is achieved by a wide range of mechanisms. Expression of all class-IA PI3K isoforms was readily detected in these cell lines. A range of genetic changes in different components of the PI3K pathway was seen in different lines. Coding variants or amplification were identified in the PIK3CA gene, and amplification of the PK3CG gene was common. Deletions in the PIK3R1 and PIK3R2 regulatory subunits were also relatively common. Notably, no genetic variants were seen in the PIK3CD gene despite p110δ being expressed in many of the lines. Genetic variants were detected in a number of genes that encode phosphatases regulating the PI3K signalling, with reductions in copy number common in PTEN, INPP4B, INPP5J, PHLLP1 and PHLLP2 genes. While the pan-PI3K inhibitor ZSTK474 attenuated cell growth in all the lines tested, isoform-selective inhibition of p110α and p110δ inhibited cell growth in only a subset of the lines and the inhibition was only partial. This suggests that functional redundancy exists between PI3K isoforms. Furthermore, while ZSTK474 was initially effective in melanoma cells with induced resistance to vemurafenib, a subset of these cell lines concurrently developed partial resistance to PI3K inhibition. Importantly, mTOR-selective or mTOR/PI3K dual inhibitors effectively inhibited cell growth in all the lines, including those already resistant to BRAF inhibitors and ZSTK474. Conclusions Overall, this indicates a high degree of diversity in the way the PI3K pathway is activated in different melanoma cell lines and that mTOR is the most effective point for targeting the growth via the PI3K pathway across all of these cell lines.

Rejuvenated ageing mesenchymal stem cells by stepwise preconditioning ameliorates surgery-induced osteoarthritis in rabbits

Aims Ageing-related incompetence becomes a major hurdle for the clinical translation of adult stem cells in the treatment of osteoarthritis (OA). This study aims to investigate the effect of stepwise preconditioning on cellular behaviours in human mesenchymal stem cells (hMSCs) from ageing patients, and to verify their therapeutic effect in an OA animal model. Methods Mesenchymal stem cells (MSCs) were isolated from ageing patients and preconditioned with chondrogenic differentiation medium, followed by normal growth medium. Cellular assays including Bromodeoxyuridine / 5-bromo-2'-deoxyuridine (BrdU), quantitative polymerase chain reaction (q-PCR), β-Gal, Rosette forming, and histological staining were compared in the manipulated human mesenchymal stem cells (hM-MSCs) and their controls. The anterior cruciate ligament transection (ACLT) rabbit models were locally injected with two millions, four millions, or eight millions of hM-MSCs or phosphate-buffered saline (PBS). Osteoarthritis Research Society International (OARSI) scoring was performed to measure the pathological changes in the affected joints after staining. Micro-CT analysis was conducted to determine the microstructural changes in subchondral bone. Results Stepwise preconditioning approach significantly enhanced the proliferation and chondrogenic potential of ageing hMSCs at early passage. Interestingly, remarkably lower immunogenicity and senescence was also found in hM-MSCs. Data from animal studies showed cartilage damage was retarded and subchondral bone remodelling was prevented by the treatment of preconditioned MSCs. The therapeutic effect depended on the number of cells applied to animals, with the best effect observed when treated with eight millions of hM-MSCs. Conclusion This study demonstrated a reliable and feasible stepwise preconditioning strategy to improve the safety and efficacy of ageing MSCs for the prevention of OA development. Cite this article: Bone Joint Res 2021;10(1):10–21.