Scalable in vitro production of defined mouse erythroblasts

Mouse embryonic stem cells (mESCs) can be manipulated in vitro to recapitulate the process of erythropoiesis, during which multipotent cells undergo lineage specification, differentiation and maturation to produce erythroid cells. Although useful for identifying specific progenitors and precursors, this system has not been fully exploited as a source of cells to analyse erythropoiesis. Here, we establish a protocol in which characterised erythroblasts can be isolated in a scalable manner from differentiated embryoid bodies (EBs). Using transcriptional and epigenetic analysis, we demonstrate that this system faithfully recapitulates normal primitive erythropoiesis and fully reproduces the effects of natural and engineered mutations seen in primary cells obtained from mouse models. We anticipate this system to be of great value in reducing the time and costs of generating and maintaining mouse lines in a number of research scenarios.

P-TEFb is degraded by Siah1/2 in quiescent cells

P-TEFb, composed of CycT1 and CDK9, regulates the elongation of transcription by RNA polymerase II. In proliferating cells, it is regulated by 7SK snRNA in the 7SK snRNP complex. In resting cells, P-TEFb is absent, because CycT1 is dephosphorylated, released from CDK9 and rapidly degraded. In this study, we identified the mechanism of this degradation. We mapped the ubiquitination and degradation of free CycT1 to its N-terminal region from positions 1 to 280. This region is ubiquitinated at six lysines, where E3 ligases Siah1 and Siah2 bind and degrade these sequences. Importantly, the inhibition of Siah1/2 rescued the expression of free CycT1 in proliferating as well as resting primary cells. We conclude that Siah1/2 are the E3 ligases that bind and degrade the dissociated CycT1 in resting, terminally differentiated, anergic and/or exhausted cells.

PCBP1 Deficient Pigs Hold the Potential to Inhibit CSFV Infection

Classical swine fever virus (CSFV), pathogen of classic swine fever, has caused severe economic losses worldwide. Poly (rC)-binding protein 1 (PCBP1), interacting with Npro of CSFV, plays a vital role in CSFV growth. Here, our research is the first report to generate PCBP1 knockout pigs via gene editing technology. The PCBP1 knockout pigs exhibited normal birth weight, reproductive-performance traits, and developed normally. Viral challenge results indicated that primary cells isolated from F0 and F1 generation pigs could significantly reduce CSFV infection. Additional mechanism exploration further confirmed that PCBP1 KO mediated antiviral effect is related with the activation of type I interferon. Beyond showing that gene editing strategy can be used to generate PCBP1 KO pigs, our study introduces a valuable animal model for further investigating infection mechanisms of CSFV that help to develop better antiviral solution.

Generation and characterization of human Fetal membrane and Decidual cell lines for reproductive biology experiments

Human fetal membrane and maternal decidua parietalis form one of the major feto-maternal interfaces during pregnancy. Studies on this feto-maternal interface is limited as several investigators have limited access to the placenta, and experience difficulties to isolate and maintain primary cells. Many cell lines that are currently available do not have the characteristics or properties of their primary cells of origin. Therefore, we created, characterized the immortalized cells from primary isolates from fetal membrane-derived amnion epithelial cells, amnion and chorion mesenchymal cells, chorion trophoblast cells and maternal decidua parietalis cells. Primary cells were isolated from a healthy full-term, not in labor placenta. Primary cells were immortalized using either a HPV16E6E7 retroviral or a SV40T lentiviral system. The immortalized cells were characterized for the morphology, cell type-specific markers, and cell signalling pathway activation. Genomic stability of these cells was tested using RNA seq, karyotyping, and short tandem repeats DNA analysis. Immortalized cells show their characteristic morphology, and express respective epithelial, mesenchymal and decidual markers similar to that of primary cells. Gene expression of immortalized and primary cells were highly correlated (R = 0.798 to R = 0.974). Short tandem repeats DNA analysis showed in the late passage number (>P30) of cell lines matched 84-100% to the early passage number (<P10) of the cell lines revealing there were no genetic drift over the passages. Karyotyping also revealed no chromosomal anomalies. Creation of these cell lines can standardize experimental approaches, eliminate subject to subject variabilities, and benefit the reproductive biological studies on pregnancies by using these cells.

SARS-CoV-2 Spike Expression at the Surface of Infected Primary Human Airway Epithelial Cells

Different serological assays were rapidly generated to study humoral responses against the SARS-CoV-2 Spike glycoprotein. Due to the intrinsic difficulty of working with SARS-CoV-2 authentic virus, most serological assays use recombinant forms of the Spike glycoprotein or its receptor binding domain (RBD). Cell-based assays expressing different forms of the Spike, as well as pseudoviral assays, are also widely used. To evaluate whether these assays recapitulate findings generated when the Spike is expressed in its physiological context (at the surface of the infected primary cells), we developed an intracellular staining against the SARS-CoV-2 nucleocapsid (N) to distinguish infected from uninfected cells. Human airway epithelial cells (pAECs) were infected with authentic SARS-CoV-2 D614G or Alpha variants. We observed robust cell-surface expression of the SARS-CoV-2 Spike at the surface of the infected pAECs using the conformational-independent anti-S2 CV3-25 antibody. The infected cells were also readily recognized by plasma from convalescent and vaccinated individuals and correlated with several serological assays. This suggests that the antigenicity of the Spike present at the surface of the infected primary cells is maintained in serological assays involving expression of the native full-length Spike.

SARS-CoV-2 spike engagement of ACE2 primes S2′ site cleavage and fusion initiation

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has resulted in tremendous loss worldwide. Although viral spike (S) protein binding of angiotensin-converting enzyme 2 (ACE2) has been established, the functional consequences of the initial receptor binding and the stepwise fusion process are not clear. By utilizing a cell–cell fusion system, in complement with a pseudoviral infection model, we found that the spike engagement of ACE2 primed the generation of S2′ fragments in target cells, a key proteolytic event coupled with spike-mediated membrane fusion. Mutagenesis of an S2′ cleavage site at the arginine (R) 815, but not an S2 cleavage site at arginine 685, was sufficient to prevent subsequent syncytia formation and infection in a variety of cell lines and primary cells isolated from human ACE2 knock-in mice. The requirement for S2′ cleavage at the R815 site was also broadly shared by other SARS-CoV-2 spike variants, such as the Alpha, Beta, and Delta variants of concern. Thus, our study highlights an essential role for host receptor engagement and the key residue of spike for proteolytic activation, and uncovers a targetable mechanism for host cell infection by SARS-CoV-2.

Construction of a Fibroblast-Associated Tumor Spheroid Model Based on a Collagen Drop Array Chip

Spheroid, a 3D aggregate of tumor cells in a spherical shape, has overcome the limitations of conventional 3D cell models to accurately mimic the in-vivo environment of a human body. The spheroids are cultured with other primary cells and embedded in collagen drops using hang drop plates and low-attachment well plates to construct a spheroid–hydrogel model that better mimics the cell–cell and cell–extracellular matrix (ECM) interactions. However, the conventional methods of culturing and embedding spheroids into ECM have several shortcomings. The procedure of transferring a single spheroid at a time by manual pipetting results in well-to-well variation and even loss or damage of the spheroid. Based on the previously introduced droplet contact-based spheroid transfer technique, we present a poly(dimethylsiloxane) and resin-based drop array chip and a pillar array chip with alignment stoppers, which enhances the alignment between the chips for uniform placement of spheroids. This method allows the facile and stable transfer of the spheroid array and even eliminates the need for a stereomicroscope while handling the cell models. The novel platform demonstrates a homogeneous and time-efficient construction and diverse analysis of an array of fibroblast-associated glioblastoma multiforme spheroids that are embedded in collagen.

Interplay between PML NBs and HIRA for H3.3 deposition on transcriptionally active interferon-stimulated genes

Promyelocytic Leukemia Nuclear Bodies (PML NBs) are nuclear membrane-less organelles physically associated with chromatin underscoring their crucial role in genome function. The H3.3 histone chaperone complex HIRA accumulates in PML NBs upon senescence, viral infection or IFN-I treatment in primary cells. Yet, the molecular mechanisms of this partitioning and its function in regulating histone dynamics have remained elusive. Here, by using specific siRNAs and protein Affimers, we identify intermolecular SUMO-SIM interactions as an essential mechanism for HIRA recruitment in PML NBs. In addition, we demonstrate that HIRA localization in the nuclear bodies is intimately linked to the presence of a soluble pool of H3.3-H4 dimers inside PML NBs, that is not found in cancer cells. Transcription inhibition prevents HIRA accumulation in PML NBs underscoring the importance of transcriptional activity to drive HIRA through PML NBs. Finally, in the context of inflammatory responses, HIRA and PML are necessary for the prolonged H3.3 deposition at the transcriptional end sites of interferon-stimulated genes (ISGs), well beyond the peak of transcription. We thus propose that HIRA partitioning in PML NBs is essential to regulate H3.3 deposition on transcriptionally active regions.

A new automated tool to quantify nucleoid distribution within mitochondrial networks

Mitochondrial DNA (mtDNA) maintenance is essential to sustain a functionally healthy population of mitochondria within cells. Proper mtDNA replication and distribution within mitochondrial networks are essential to maintain mitochondrial homeostasis. However, the fundamental basis of mtDNA segregation and distribution within mitochondrial networks is still unclear. To address these questions, we developed an algorithm, Mitomate tracker to unravel the global distribution of nucleoids within mitochondria. Using this tool, we decipher the semi-regular spacing of nucleoids across mitochondrial networks. Furthermore, we show that mitochondrial fission actively regulates mtDNA distribution by controlling the distribution of nucleoids within mitochondrial networks. Specifically, we found that primary cells bearing disease-associated mutations in the fission proteins DRP1 and MYH14 show altered nucleoid distribution, and acute enrichment of enlarged nucleoids near the nucleus. Further analysis suggests that the altered nucleoid distribution observed in the fission mutants is the result of both changes in network structure and nucleoid density. Thus, our study provides novel insights into the role of mitochondria fission in nucleoid distribution and the understanding of diseases caused by fission defects.

Establishment of an Efficient Immortalization Strategy Using HMEJ-Based bTERT Insertion for Bovine Cells

Immortalized cell lines have been used in a wide range of applications in research on immune disorders and cellular metabolic regulation due to the stability and uniformity of their cellular characteristics. At present, the investigation into molecular functions and signaling pathways within bovine cells remains largely limited by the lack of immortalized model cells. Current methods for immortalizing bovine cells are mainly restricted to the ectopic expression of human telomerase reverse transcriptase (hTERT) through transient transfection or virus-mediated delivery, which have defects in efficiency and reliability. In this study, we identified bovine TERT (bTERT) as a novel potent biofactor for immortalizing bovine cells with great advantages over hTERT, and established an efficient and easily manipulated strategy for the immortalization of bovine primary cells. Through the homology-mediated end-joining-based insertion of bTERT at the ROSA26 locus, we successfully generated immortalized bovine fetal fibroblast cell lines with stable characteristics. The observed limitation of this strategy in immortalizing bovine bone marrow-derived macrophages was attributed to the post-translational modification of bTERT, causing inhibited nuclear localization and depressed activity of bTERT in this terminally differentiated cell. In summary, we constructed an innovative method to achieve the high-quality immortalization of bovine primary cells, thereby expanding the prospects for the future application of immortalized bovine model cell lines.