Large-cohort humanized NPI mice reconstituted with CD34+ hematopoietic stem cells are feasible for evaluating preclinical cancer immunotherapy.

Cancer immunotherapy has achieved impressive therapeutic effects in many cancers, while only a small subset of patients benefit from it and some patients even have experienced severe toxicity. It is urgent to develop a feasible large-cohort humanized mouse model to evaluate the pre-clinical efficacy and safety of cancer immunotherapy. Furthermore, developing potentially effective combination therapy between cancer immunotherapy and other therapies also needs humanized mouse model to adequately mimic clinical actual setting. Herein, we established a humanized mouse model engrafted with less human CD34+ HSCs than ever before and then evaluated reconstitution efficiency and the profiles of human immune cells in this humanized mouse model. Also, this humanized mouse model was used to evaluate the preclinical efficacy and safety of cancer immunotherapy. For each batch of CD34+ HSCs humanized mouse model, a relatively-large cohort with over 25% human CD45+ cells in peripheral blood was established. This humanized mouse model could efficiently reconstitute human innate and adaptive immune cells. This humanized mouse model supported patient-derived xenograft tumor growth and tumor infiltration of PD-1+ human T cells. Furthermore, therapeutic efficacy, re-activation of tumor-infiltrated T cells, and side effects of checkpoint blockade therapy could be monitored in this humanized mouse model. Human T cells from this humanized mouse model were successfully engineered with CD19-CAR. CD19 CAR-T cells could effectively deplete B cells and suppress tumor growth of acute lymphoblastic leukemia in vivo in this humanized mouse model. This humanized mouse model also could be used to demonstrate the efficacy of bispecific antibodies, such as anti-CD19/CD3. Overall, our work provides a feasible large-cohort humanized mouse model for evaluating a variety of cancer immunotherapy approaches including checkpoint inhibitors, adoptive cell therapy, and bispecific antibody therapy, and demonstrates that human T cells from this humanized mouse model possess anti-tumor activities in vitro and in vivo.

The plant hormone abscisic acid stimulates megakaryocyte differentiation from human iPSCs in vitro.

In the clinic, the supply of platelets is frequently insufficient to meet transfusion needs. To address this issue, many scientists have established the derivation of functional platelets from CD34+ cells or human pluripotent stem cells (PSCs). However, the yield of platelets is still far below what is required. Here we found that the plant hormone abscisic acid (ABA) could increase the generation of megakaryocytes (MKs) and platelets from human induced PSCs (hiPSCs). During platelet derivation, ABA treatment promoted the generation of CD34+/CD45+ HPCs and CD41+ MKs on day 14 and then increased CD41+/CD42b+ MKs and platelets on day 19. Moreover, we found ABA-mediated activation of Akt and ERK1/2 signal pathway through receptors LANCL2 and GRP78 in a PKA-dependent manner on CD34+/CD45+ cells. In conclusion, our data suggest that ABA treatment can promote CD34+/CD45+ HPC proliferation and CD41+ MK differentiation.

Low Platelet Counts at Diagnosis Predict Better Survival for Patients with Intermediate-Risk Acute Myeloid Leukemia.

BACKGROUND: Aggressive growth of primitive and immature cells in the bone marrow results in reductions in megakaryocyte and platelet (PLT) counts, leading to thrombocytopenia in acute myeloid leukemia (AML). However, not all AML patients show thrombocytopenia at the time of diagnosis, and the association of PLT count with patient survival is largely unknown. METHODS: A retrospective study was performed to determine PLT counts at diagnosis in the peripheral blood in 291 newly diagnosed AML patients and assess the association of PLT counts with the overall survival (OS) and disease-free survival (DFS) of these patients. RESULTS: Low PLT counts (≤40 × 109/L) were associated with better outcomes for the whole cohort (5-year OS, 55.1 ± 3.8 vs. 35.3 ± 3.5%, p < 0.001; 5-year DFS, 49.1 ± 3.8 vs. 25.7 ± 4.0%, p < 0.001) and intermediate-risk patients (5-year OS, 64.5 ± 5.4 vs. 41.0 ± 4.8%, p < 0.001; 5-year DFS, 60.8 ± 5.6 vs. 28.6 ± 5.6%, p < 0.001). Moreover, low PLT counts were related to deeper molecular remission. Low PLT counts correlated with better survival of intermediate-risk AML patients treated with chemotherapy only. Allogeneic hematopoietic stem cell transplantation attenuated the negative impact of high PLT counts on the survival of intermediate-risk patients. Furthermore, univariate and multivariate analyses demonstrated that PLT count at diagnosis was an independent prognostic factor for intermediate-risk AML. CONCLUSION: PLT count at diagnosis predicts survival for patients with intermediate-risk AML.

Circulating CXCR5+CD4+ T cells assist in the survival and growth of primary diffuse large B cell lymphoma cells through interleukin 10 pathway.

Diffuse large B cell lymphoma (DLBCL) is a common and aggressive cancer caused by the malignant transformation of B cells. Although it has been established that the follicular helper T (Tfh) cells play a central role in B cell development, little information is available on their involvement in DLBCL pathogenesis. We studied the role of the peripheral Tfh equivalent, the CXCR5+ CD4+ T cells, in DLBCL. Data showed that compared to CXCR5- CD4+ T cells, CXCR5+ CD4+ T cells were significantly more effective at promoting the proliferation as well as inhibiting the apoptosis of primary autologous DLBCL tumor cells. Surprisingly, we found that at equal cell numbers, CXCR5+ CD4+ T cells in DLBCL patients secreted significantly less interleukin (IL)-21 than CXCR5- CD4+ T cells, while the level of IL-10 secretion was significant elevated in the CXCR5+ compartment compared to the CXCR5- compartment. Neutralization of IL-10 in the primary DLBCL-CXCR5+ CD4+ T cell coculture compromised the CXCR5+ CD4+ T cell-mediated pro-tumor effects, in a manner that was dependent on the concentration of anti-IL-10 antibodies. The CXCR5+ compartment also contained significantly lower frequencies of cytotoxic CD4+ T cells than the CXCR5- compartment. In conclusion, our investigations discovered a previously unknown pro-tumor role of CXCR5-expressing circulating CD4+ T cells, which assisted the survival and proliferation of primary DLBCL cells through IL-10.

Adaptive B cell responses in rituximab-treated diffuse large B cell lymphoma patients during complete remission.

Rituximab is a chimeric monoclonal antibody directed against the CD20 antigen. Treatment using rituximab in combination with chemotherapy has dramatically improved overall survival rate of diffuse large B cell lymphoma (DLBCL). Since rituximab can deplete both lymphoma B cells and normal B cells, how rituximab-treatment affects normal B cell function in DLBCL patients under remission is unclear. Here, we examined peripheral blood B cell composition and antigen-specific B cell responses in DLBCL patients in remission and observed reductions in the frequencies of total B cell as well as several major B cell subsets, including CD19(+)IgD(+) naive B cells, CD19(+)IgD(-)CD27(+) memory B cells, and CD19(lo)CD27(hi) plasmablasts. Moreover, tetanus toxin (TT)-specific B cell proliferation was reduced in DLBCL patients in remission. On the other hand, HA-specific IgG-secreting B cell responses could be stimulated by influenza vaccination in DLBCL patients in remission, demonstrating that the machinery for generating de novo adaptive B cell responses was functional in DLBCL patients in remission. Our results provided insights in normal B cell function in DLBCL patients in remission.

Effects of volatile vs. propofol-based intravenous anesthetics on the alveolar inflammatory responses to one-lung ventilation: a meta-analysis of randomized controlled trials.

OBJECTIVE: The aim of this meta-analysis is to compare the potential effects of inhalation anesthetics with total intravenous anesthetics on alveolar cytokine expression and lung-related clinical outcomes in patients undergoing one-lung ventilation (OLV) for thoracic surgery. METHODS: We retrieved the PubMed, EMBASE, and the Cochrane Library respectively to identify randomized controlled trials comparing different anesthetics (volatile anesthetics vs. intravenous anesthetics) on the pulmonary inflammatory response to OLV. The primary outcomes were the levels of alveolar concentrations of inflammatory cytokines. RESULTS: Eight randomized controlled trials that included 365 patients were screened. Overall, there were significant differences in the concentration of alveolar inflammatory mediators between volatile group and intravenous group, in which volatile group had lower levels of TNF-α (SMD -1.51; 95 % CI -2.15 to -0.87; p < 0.001), IL-6 (SMD -0.70; 95 % CI -0.99 to -0.41; p < 0.001) and IL-8 (SMD -1.32; 95 % CI -2.20 to -0.45; p = 0.003). The overall number of pulmonary complications in the volatile group was smaller (RR 0.42; 95 % CI 0.23-0.77; p = 0.005) and patients in that group had significantly abridged hospitalization stay (WMD -3.59 days; 95 % CI -5.70 to -1.48 days; p = 0.001). CONCLUSIONS: Inhalation anesthetics might be preferable in patients undergoing OLV for thoracic surgery and their protective effects might work via attenuating inflammatory responses.

Alterations of circulating follicular helper T cells and interleukin 21 in diffuse large B-cell lymphoma.

CD4+ CXCR5+ T cell in peripheral blood is known as circulating follicular helper T cell (Tfh), which can produce interleukin 21 (IL-21). In the current study, we investigated changes of circulating Tfh and its correlation with IL-21 in diffuse large B-cell lymphoma (DLBCL). Circulating Tfh and its subtypes were detected by flow cytometry in the peripheral blood of 32 healthy donors and 62 DLBCL cases. Data demonstrated that percentage of circulating Tfh in CD4+ T cells was significantly increased in DLBCL (11.3 %) than in controls (8.5 %) (p = 0.001). Studying the subtypes of Tfh revealed that the upregulation of circulating Tfh was contributed by Tfh-Th2 subtype and Tfh-Th17 subtype. Also, we identified that prevalence of Tfh was significantly elevated in cases with advanced stages (stages III and IV). Interestingly, the elevation of circulating Tfh was negatively correlated with serum IL-21 in DLBCL patients. In addition, a positive correlation between circulating Tfh and IL-21 receptor on CD + 8 T cells was observed in patients. This study suggests involvement of circulating Tfh and IL-21 in the pathogenesis and progression of DLBCL and provides a potential target for treating this disease.

Fibroblast growth factor receptor 4 polymorphisms and the prognosis of non-Hodgkin lymphoma.

Fibroblast growth factors and their receptors (FGFRs) play important roles in blood system. FGFR4 rs351855 (Gly388Arg) polymorphism has shown to be a risk factor for many diseases. The aim of this study was to investigate the association between FGFR4 polymorphisms and the susceptibility to non-Hodgkin lymphoma (NHL) in the Chinese population. We identified two polymorphisms in the FGFR4 gene, rs351855G/A (Gly388Arg), and rs147603016G/A, by polymerase chain reaction-restriction fragment length polymorphism in 412 NHL cases and 476 healthy controls. Results showed that frequencies of AA genotype and A allele in rs351855 (Gly388Arg) polymorphism were significantly higher in patients than in controls (odds ratio (OR) 2.12, 95 % confidence intervals (CI) 1.99-3.48, P < 0.0001; OR 1.45, 95 % CI 1.21-1.88, P < 0.0001, respectively; data were adjusted for age and sex). The rs147603016G/A polymorphism did not show any correlation with NHL. When analyzing the survival time of NHL patients with FGFR4 rs351855G/A polymorphism, cases with AA genotype had significantly shorter survival time compared to the patients with GG and GA genotypes (P = 0.002). These results suggested polymorphism in FGFR4 gene was associated with increased susceptibility to NHL and could be used as a prognostic marker for this malignancy.

Hematopoietic stem cell regeneration enhanced by ectopic expression of ROS-detoxifying enzymes in transplant mice.

High levels of reactive oxygen species (ROS) can exhaust hematopoietic stem cells (HSCs). Thus, maintaining a low state of redox in HSCs by modulating ROS-detoxifying enzymes may augment the regeneration potential of HSCs. Our results show that basal expression of manganese superoxide dismutase (MnSOD) and catalase were at low levels in long-term and short-term repopulating HSCs, and administration of a MnSOD plasmid and lipofectin complex (MnSOD-PL) conferred radiation protection on irradiated recipient mice. To assess the intrinsic role of elevated MnSOD or catalase in HSCs and hematopoietic progenitor cells, the MnSOD or catalase gene was overexpressed in mouse hematopoietic cells via retroviral transduction. The impact of MnSOD and catalase on hematopoietic progenitor cells was mild, as measured by colony-forming units (CFUs). However, overexpressed catalase had a significant beneficial effect on long-term engraftment of transplanted HSCs, and this effect was further enhanced after an insult of low-dose γ-irradiation in the transplant mice. In contrast, overexpressed MnSOD exhibited an insignificant effect on long-term engraftment of transplanted HSCs, but had a significant beneficial effect after an insult of sublethal irradiation. Taken together, these results demonstrate that HSC function can be enhanced by ectopic expression of ROS-detoxifying enzymes, especially after radiation exposure in vivo.

Association of peripheral CD4+ CXCR5+ T cells with chronic lymphocytic leukemia.

Accumulating evidences indicate that immune dysregulation plays a key role in both lymphomagenesis and patient outcome of chronic lymphocytic leukemia (CLL). Peripheral blood CD4+ CXCR5+ T cells, known as circulating follicular helper T cells (Tfh), can induce B cell activation and production of specific antibody responses. The aim of the study was to investigate changes of circulating Tfh in CLL. Tfh and it subtypes were tested by measuring CD4, CXCR5, CXCR3, and CCR6 in 72 CLL cases and 86 healthy controls using flow cytometry. Data showed that the percentage of Tfh in the peripheral CD4+ T cells was significantly increased in CLL (25.1%) than in controls (8.4%) (p < 0.001). Further analysis revealed that the upregulation of Tfh was contributed by Tfh-th2 subtype and Tfh-th17 subtype. Investigating staging of the cases demonstrated that the prevalence of Tfh was significantly elevated in cases with Binet stage C (37.3%) than those with stage A (20.1 %) or stage B (23.9 %). In addition, we analyzed Tfh in patients with immunoglobulin variable heavy chain (IGHV) gene mutational status. Results presented that Tfh-th17 subtype had clearly higher frequency in patients with IGHV mutation compared to the unmutated cases (p = 0.035). This study suggested the involvement of Tfh in the pathogenesis and progression of CLL, and provided a potential target for treating this disease.

Whole transcriptome analysis of platelet concentrates during storage.

BACKGROUND: Platelets are anucleated blood cells and contain various RNA species. We investigated the changes in the whole transcriptome expression profile of platelet concentrates (PC) during storage to explore biological functions and biomarkers in platelet storage damage. MATERIALS AND METHODS: Platelets were collected by apheresis from eight healthy blood donors and stored from day 0 to day 4. Platelet phenotyping and function analysis were used to detect platelet activity during storage. RNA-sequencing was used to detect changes in expression of mRNA, lncRNA and circRNA in the PC during storage. Gene ontology and KEGG analyses were applied to predict the functional distribution of differential expression of mRNA. Gene set enrichment analysis was used to analyze the differential levels of gene pathways. Finally, polymerase chain reaction (PCR) analysis was performed to verify the expression of three mRNA (POLE2, DCUN1D4, DAD1). RESULTS: In total, 10,767 mRNA, 2,923 lncRNA and 68,550 circRNA were detected in the PC by RNA-sequencing. The expression levels of 222 mRNA changed significantly from day 0 to day 4 of storage: 58 increased continuously and 145 decreased continuously. Differentially expressed mRNA may be involved in physiological processes such as platelet activation, platelet aggregation, endocytosis, and apoptosis. Expression levels of 1,413 lncRNA were obvious. The levels of 42 species increased and the levels of 28 species decreased. The expression levels of 198 species of circRNA changed significantly, with those of 13 species changing continuously. The differential levels of expression of DAD1, DCUN1D4 and POLE1 mRNA, shown by RNA sequencing, were validated by PCR assay. DISCUSSION: Changes in mRNA, lncRNA and circRNA during platelet storage may be closely related to platelet apoptosis and physiological functions in the platelet storage lesion. The expression levels of DAD1, DCUN1D4 and POLE1 could be biomarkers to monitor platelet status in PC bags.

Potential benefits, mechanisms, and uncertainties of convalescent plasma therapy for COVID-19.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China led to a public health emergency of international concern, putting all health organizations on high alert in the beginning of 2020. Corona virus disease 2019 (COVID-19) is highly infectious and has resulted in thousands of deaths which exceeded that of the SARS coronavirus (SARS-CoV) outbreak back in 2002 and 2003 in China. Besides, the number of diagnosed patients, patients who are suspected to have contracted the disease, and deaths are increasing worldwide. Unfortunately, effective drugs and vaccines to combat SARS-CoV-2 are still lacking. Convalescent plasma, a seemingly successful treatment for COVID-19 patients, proved to be of huge value in terms of saving severely ill patients. This review introduces the reported effects, potential mechanisms, and future uncertainties of convalescent plasma therapy in the treatment of COVID-19 patients, in the hopes that it will provide useful information for relevant physicians and researchers.

Diagnosis of paroxysmal nocturnal hemoglobinuria with flowcytometry panels including CD157: Data from the real world.

BACKGROUND: Several studies have used CD157 in white blood cells with or without proaerolysin (fluorescein-labeled proaerolysin [FLAER])-based flow cytometry assays in the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). METHODS: We designed a seven-color CD marker panel comprising FLAER, CD15, CD64, CD24, CD14, CD157, and CD45 to verify CD157's clinical applicability and diagnostic performance in a clinical setting. RESULTS: A total of 356 samples were tested. These included 43 PNH-positive samples and 313 PNH-negative samples. PNH clones confirmed by the CD157/FLAER combination were almost identical in size to the clones detected by the CD24/CD14/FLAER combination, and the accuracy of the CD157/FLAER combination was 100% in granulocytes and 99.7% in monocytes. Substitution of FLAER with CD157 resulted in 1.9% and 3.5% false-positives in granulocytes and monocytes, respectively. The accuracy was 98.3% and 96.9% in granulocytes and monocytes, respectively. Moreover, the loss of CD157 expression in granulocytes and monocytes was commonly observed in non-PNH patients. Some monocytes in non-PNH patients had weak expression of CD14 but normal expression of FLAER. In this study, PNH clones in granulocytes were always lower than those in matched monocytes. CONCLUSIONS: We performed the first prospective exploration of the clinical usefulness of FLAER and CD157 in simultaneously recognizing PNH clones in granulocytes and monocytes and verified the applicability of CD157 in substitute for both CD14 and CD24. In the conditions where FLAER is not available, substitution of FLAER with CD157 is acceptable for the identification of PNH clones under the premise of giving full attention to the potential for false-positives.

Absence of cyclin-dependent kinase inhibitor p27 or p18 increases efficiency of iPSC generation without induction of iPSC genomic instability.

Mechanisms underlying the generation of induced pluripotent stem cells (iPSC) and keeping iPSC stability remain to be further defined. Accumulated evidences showed that iPSC reprogramming may be controlled by the cell-division-rate-dependent model. Here we reported effects of absence of mouse p27 or p18 on iPSC generation efficiency and genomic stability. Expression levels of cyclin-dependent kinases inhibitors (CDKIs), p21, p27, and p18 decreased during iPSC reprogramming. Like p21 loss, p27 or p18 deficiency significantly promoted efficiency of iPSC generation, whereas ectopic expression of p27, p18, or treatment with CDK2 or CDK4 inhibitors repressed the reprogramming rate, suggesting that CDKIs-regulated iPSC reprogramming is directly related with their functions as CDK inhibitors. However, unlike p21 deletion, absence of p27 or p18 did not increase DNA damage or chromosomal aberrations during iPSC reprogramming and at iPSC stage. Our data not only support that cell cycle regulation is critical for iPSC reprogramming, but also reveal the distinction of CDKIs in somatic cell reprogramming.

Optimizing the method for generation of integration-free induced pluripotent stem cells from human peripheral blood.

BACKGROUND: Generation of induced pluripotent stem cells (iPSCs) from human peripheral blood provides a convenient and low-invasive way to obtain patient-specific iPSCs. The episomal vector is one of the best approaches for reprogramming somatic cells to pluripotent status because of its simplicity and affordability. However, the efficiency of episomal vector reprogramming of adult peripheral blood cells is relatively low compared with cord blood and bone marrow cells. METHODS: In the present study, integration-free human iPSCs derived from peripheral blood were established via episomal technology. We optimized mononuclear cell isolation and cultivation, episomal vector promoters, and a combination of transcriptional factors to improve reprogramming efficiency. RESULTS: Here, we improved the generation efficiency of integration-free iPSCs from human peripheral blood mononuclear cells by optimizing the method of isolating mononuclear cells from peripheral blood, by modifying the integration of culture medium, and by adjusting the duration of culture time and the combination of different episomal vectors. CONCLUSIONS: With this optimized protocol, a valuable asset for banking patient-specific iPSCs has been established.

The prevalence and function of CD4+CXCR5+Foxp3+ follicular regulatory T cells in diffuse large B cell lymphoma.

CD4+CXCR5+Foxp3+ follicular regulatory T (Tfr) cells possess critical roles in suppressing the germinal center reaction, B cell activation, and follicular helper T cell (Tfh) cytokine secretion. Since diffuse large B cell lymphoma (DLBCL) can arise from B cells undergoing germinal center reaction and/or differentiation, we hypothesized that Tfr cells might be involved in DLBCL. In the present study, we recruited thirty-five DLBCL patients and twenty-five healthy controls. Data showed that DLBCL patients presented an enrichment of circulating CD4+CXCR5+Foxp3+ Tfr cells compared to controls. In the primary tumor isolated from enlarged lymph nodes, Tfr cells made up of roughly 3% to 16% of infiltrating T cells. Higher levels of tumor-infiltrating Tfr cells were observed in patients with less advanced DLBCL stages, and in patients that stayed in remission 24 months after the initial R-CHOP treatment. High BCL6 and high FOXP3 expression was observed in Tfr cells ex vivo. After anti-CD3/CD28 and IL-2 stimulation, the Tfr cells more closely resembled Treg cells and presented high IL10 and TGFB1 expression. CD4+CD25+CXCR5+ Tfr cells and CD4+CD25+CXCR5- non-Tfr Treg cells could suppress CD4+CD25- Tconv cell and CD8+ T cell proliferation with similar capacity. However, Tfr cells were less capable of suppressing IFNG expression than Treg cells, and although both cell types supported CD19+ tumor cell proliferation, Tfr cells were less supportive than the non-Tfr Treg cells. Overall, this study suggested that Tfr cells were involved in intratumoral immunity, were likely beneficial to DLBCL patients, and were functionally distinctive from non-Tfr Treg cells. The distribution pattern and the prognostic value of Tfr cells in DLBCL should be examined in further studies.

Targeted genome engineering in human induced pluripotent stem cells from patients with hemophilia B using the CRISPR-Cas9 system.

BACKGROUND: Replacement therapy for hemophilia remains a lifelong treatment. Only gene therapy can cure hemophilia at a fundamental level. The clustered regularly interspaced short palindromic repeats-CRISPR associated nuclease 9 (CRISPR-Cas9) system is a versatile and convenient genome editing tool which can be applied to gene therapy for hemophilia. METHODS: A patient's induced pluripotent stem cells (iPSCs) were generated from their peripheral blood mononuclear cells (PBMNCs) using episomal vectors. The AAVS1-Cas9-sgRNA plasmid which targets the AAVS1 locus and the AAVS1-EF1α-F9 cDNA-puromycin donor plasmid were constructed, and they were electroporated into the iPSCs. When insertion of F9 cDNA into the AAVS1 locus was confirmed, whole genome sequencing (WGS) was carried out to detect the off-target issue. The iPSCs were then differentiated into hepatocytes, and human factor IX (hFIX) antigen and activity were measured in the culture supernatant. Finally, the hepatocytes were transplanted into non-obese diabetic/severe combined immunodeficiency disease (NOD/SCID) mice through splenic injection. RESULTS: The patient's iPSCs were generated from PBMNCs. Human full-length F9 cDNA was inserted into the AAVS1 locus of iPSCs of a hemophilia B patient using the CRISPR-Cas9 system. No off-target mutations were detected by WGS. The hepatocytes differentiated from the inserted iPSCs could secrete hFIX stably and had the ability to be transplanted into the NOD/SCID mice in the short term. CONCLUSIONS: PBMNCs are good somatic cell choices for generating iPSCs from hemophilia patients. The iPSC technique is a good tool for genetic therapy for human hereditary diseases. CRISPR-Cas9 is versatile, convenient, and safe to be used in iPSCs with low off-target effects. Our research offers new approaches for clinical gene therapy for hemophilia.

Factors Affecting Mouse Somatic Cell Nuclear Reprogramming by Rabbit Ooplasms.

Successful development of interspecies somatic cell nuclear transfer (iSCNT) embryos depends on compatibilities between ooplasmic and nuclear components. However, the mechanisms by which the compatibilities are regulated are still unknown. In this study, using mouse Oct4-green fluorescent protein (GFP) cells as donors and rabbit oocytes as recipients, we show that Oct4 and other pluripotency related genes were reactivated in some of mouse-rabbit iSCNT embryos, which could also activate Oct4 promoter-driven GFP reporter gene expression. Series nuclear transfer improved the efficiency of Oct4 reactivation. DNA demethylation of Oct4 promoter was detected in GFP positive iSCNT blastocysts, whereas GFP negative iSCNT embryos showed a low efficiency. Our results demonstrate that Oct4-GFP can well label the embryos with epigenetic remodeling and reactivation of pluripotent gene expression. Abundant rabbit mitochondria specific DNAs were identified in reconstructed mouse-rabbit embryos throughout preimplantation stages. Our data demonstrate that epigenetic remodeling and the complete mitochondrial match are not necessary for successful iSCNT embryo development before implantation.

IL-22 Impedes the Proliferation of Schwann cells: Transcriptome Sequencing and Bioinformatics Analysis.

Schwann cells (SCs) proliferation is crucial for nerve regeneration following nerve injury. This study aims to investigate effects of interleukin-22 (IL-22) on SCs proliferation in vitro, as well as the corresponding mechanism. Rat SCs were treated with 100 ng/ml rat IL-22 for 48 h, and cell proliferation and apoptosis were detected using fluorescent staining and flow cytometry. After transcriptome sequencing, raw reads were filtered and mapped to reference genome rn5. Then, differentially expressed genes (DEGs) and long non-coding RNAs (DElncRNAs) between IL-22 and control groups were identified (tool: Cuffdiff). Functional and pathway enrichment analyses were performed (tool: GOFunction), and protein-protein interaction (PPI) network was constructed (tool: STRING and Cytoscape). Furthermore, Pearson's correlations between DEGs and DElncRNAs were analyzed, and regulatory network of DEGs, DElncRNAs, and transcription factors (TFs) was constructed. IL-22 significantly inhibited proliferation (p value < 0.05) and promoted apoptosis of Schwann cells. Totally, 932 DEGs and 118 DElncRNAs were identified, among which Ccl2 and Ccna2 were hub genes in PPI network. Up-regulated DEGs were enriched in apoptosis related terms, whereas down-regulated DEGs were enriched in proliferation related terms. DElncRNAs like NONRATT023505, NONRATG020400, and NONRATT022748 were correlated with multiple DEGs enriched in cell cycle and division. Moreover, up-regulated TFs Egr1, Cebpd, and Atf4 play crucial roles in regulatory network, and NONRATG020400-Cebpd-Ccl2, NONRATT023505/NONRATT022748-Atf4-Ccna2, and NONRATT022748-Egr1-Id1/Aldoc/Eno2/F3/Serpine1 regulatory pathways were identified in SCs after IL-22 treatment. IL-22 might influence SCs proliferation and apoptosis via regulating lncRNA-TF-gene pathways in SCs. However, more studies are required to confirm these results.