Osteogenic differentiation from mouse adipose-derived stem cells and bone marrow stem cells.

Mesenchymal stem cells (MSCs) have been successfully cultured and proliferated in vitro and can differentiate into a variety of specific cell types, such as adipocytes or osteocytes, through chemical stimulation. One of the major applications of MSCs is in regenerative medicine research. MSCs can be collected from many adult tissues. In this experiment, an 8-week-old expresses green fluorescent protein (EGFP) transgenic mouse, FVB/NCrl-Tg(Pgk1-EGFP)01Narl, was used to obtain adipose-derived stem cells (ADSCs) from abdominal adipose tissue and bone marrow stem cells (BMSCs) from femur bone marrow. We compared the differences in the growth rate and differentiation ability of ADSCs and BMSCs. The growth curves of different generations (P1 and P3) of the stem cells showed that the proliferation rate of ADSCs was significantly higher than that of BMSCs. The purity of stem cells was measured by the number of colony-forming unit fibroblast. The results show that the number of colonies of ADSCs at different generations (P1 and P3) was significantly higher than that of BMSCs and that the purity of ADSCs was greater than that of BMSCs. Comparing the ability of ADSCs and BMSCs to induce osteogenic differentiation and the expression of Runx2 and Opn genes, the results show that ADSCs had a higher rate of osteogenic differentiation than BMSCs. In summary, mouse ADSCs display similar osteogenic differentiation ability to BMSCs but have a better capacity than BMSCs in terms of stem cell purity and cell proliferation in vitro.

Development and Efficacy of Lateral Flow Point-of-Care Testing Devices for Rapid and Mass COVID-19 Diagnosis by the Detections of SARS-CoV-2 Antigen and Anti-SARS-CoV-2 Antibodies.

The COVID-19 pandemic is an ongoing global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2020-2021. COVID-19 is becoming one of the most fatal pandemics in history and brings a huge challenge to the global healthcare system. Opportune detection, confinement, and early treatment of infected cases present the first step in combating COVID-19. Diagnosis via viral nucleic acid amplification tests (NAATs) is frequently employed and considered the standard procedure. However, with an increasing urge for point-of-care tests, rapid and cheaper immunoassays are widely utilized, such as lateral flow immunoassay (LFIA), which can be used for rapid, early, and large-scale detection of SARS-CoV-2 infection. In this narrative review, the principle and technique of LFIA applied in COVID-19 antigen and antibody detection are introduced. The diagnostic sensitivity and specificity of the commercial LFIA tests are outlined and compared. Generally, LFIA antigen tests for SARS-CoV-2 are less sensitive than viral NAATs, the "gold standard" for clinical COVID-19 diagnosis. However, antigen tests can be used for rapid and mass testing in high-risk congregate housing to quickly identify people with COVID-19, implementing infection prevention and control measures, thus preventing transmission. LFIA anti-SARS-CoV-2 antibody tests, IgM and/or IgG, known as serology tests, are used for identification if a person has previously been exposed to the virus or vaccine immunization. Notably, advanced techniques, such as LFT-based CRISPR-Cas9 and surface-enhanced Raman spectroscopy (SERS), have added new dimensions to the COVID-19 diagnosis and are also discussed in this review.

Co-cultured bone marrow mesenchymal stem cells repair thioacetamide-induced hepatocyte damage.

Adult stem cells, such as bone marrow mesenchymal stem cells (BMSCs), are postdevelopmental cells found in many bone tissues. They are capable of multipotent differentiation and have low immune-rejection characteristics. Hepatocytes may become inflamed and produce a large number of free radicals when affected by drugs, poisoning, or a viral infection. The excessive accumulation of free radicals in the extracellular matrix (ECM) eventually leads to liver fibrosis. This study aims to investigate the restorative effects of mouse bone marrow mesenchymal stem cells (mBMSCs) on thioacetamide (TAA)-induced damage in hepatocytes. An in vitro transwell co-culture system of HepG2 cells were co-cultured with mBMSCs. The effects of damage done to TAA-treated HepG2 cells were reflected in the overall cell survival, the expression of antioxidants (SOD1, GPX1, and CAT), the ECM (COL1A1 and MMP9), antiapoptosis characteristics (BCL2), and inflammation (TNF) genes. The majority of the damage done to HepG2 by TAA was significantly reduced when cells were co-cultured with mBMSCs. The signal transducer and activator of transcription 3 (STAT3) and its phosphorylated STAT3 (p-STAT3), as related to cell growth and survival, were detected in this study. The results show that STAT3 was significantly decreased in the TAA-treated HepG2 cells, but the STAT3 and p-STAT3 of HepG2 cells were significantly activated when the TAA-treated HepG2 co-cultured with mBMSCs. Strong expression of interleukin (Il6) messenger RNA in co-cultured mBMSCs/HepG2 indicated mBMSCs secret the cytokines IL-6, which promotes cell survival through downstream STAT3 activation and aid in the recovery of HepG2 cells damaged by TAA.

Loss of angiotensin converting enzyme II (ACE2) accelerates the development of liver injury induced by thioacetamide.

Angiotensin converting enzyme II (ACE2), an angiotensin converting enzyme (ACE) homologue that displays antagonist effects on ACE/angiotensin II (Ang II) axis in renin-angiotensin system (RAS), could play a protective role against liver damages. The purpose of this study is to investigate whether inflammation-mediated liver injury could be affected by ACE2 derived pathways in the RAS. Eight-weeks-old wild-type (WT; C57BL/6) and Ace2 KO (hemizygous Ace2-/y) male mice were used to induce liver fibrosis by thioacetamide (TAA) administration (0, 100, and 200 mg/kg BW). The mice administrated with TAA could be successfully induced liver fibrosis in a TAA-dose dependent manner. Compared to WT mice, the results show that Ace2 KO mice have high sensitive, and developed more serious reaction of hepatic inflammation and fibrosis by TAA administration. The physiological and pathological examinations demonstrated higher serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) levels, infiltration of white blood cells and fibrotic lesions within liver in the Ace2 KO mice. The severe liver damage of Ace2 KO mice were also confirmed by the evidence of higher expression of hepatic inflammation-related genes (IL-6 and Tnf) and fibrosis-related genes (Col1a1, Timp1 and Mmp9). Ace2 gene deficiency could lead to a severe inflammation and collagen remodeling in the liver administrated by TAA, and the responses lead the pathogenesis of liver fibrosis. Our studies provided the main messages and favorable study directions of relationship of Ace2 and liver disease.

Ability of an alkali-tolerant mutant strain of the microalga Chlorella sp. AT1 to capture carbon dioxide for increasing carbon dioxide utilization efficiency.

An alkali-tolerant Chlorella sp. AT1 mutant strain was screened by NTG mutagenesis. The strain grew well in pH 6-11 media, and the optimal pH for growth was 10. The CO2 utilization efficiencies of Chlorella sp. AT1 cultured with intermittent 10% CO2 aeration for 10, 20 and 30min at 3-h intervals were approximately 80, 42 and 30%, respectively. In alkaline medium (pH=11) with intermittent 10% CO2 aeration for 30min at 3-, 6- and 12-h intervals, the medium pH gradually changed to 10, and the biomass productivities of Chlorella sp. AT1 were 0.987, 0.848 and 0.710gL-1d-1, respectively. When Chlorella sp. AT1 was aerated with 10% CO2 intermittently for 30min at 3-h intervals in semi-continuous cultivation for 21days, the biomass concentration and biomass productivity were 4.35gL-1 and 0.726gL-1d-1, respectively. Our results show that CO2 utilization efficiency can be markedly increased by intermittent CO2 aeration and alkaline media as a CO2-capturing strategy for alkali-tolerant microalga cultivation.

Identifying the regulative role of NF-κB binding sites within promoter region of human matrix metalloproteinase 9 (mmp-9) by TNF-α induction.

Matrix metalloproteinase 9 (MMP-9), a member of MMP family, is involved in many physiological processes, including cardiovascular disease (CVD). Tumor necrosis factor-α (TNF-α) is considered a cytokine with pleiotropic biological capabilities and leads to the process of CVD when TNF-α is abnormally released and stimulates MMP-9 expression and activation. In this study, we investigated the molecular mechanism of TNF-α-regulated MMP-9 expression. The experimental results confirm that TNF-α could upregulate MMP-9 expression in heart myoblast H9c2 cells of rat. To evaluate the MMP-9 regulation at transcriptional level, a DNA fragment of 2.2 kb (-2168/+18) of human mmp-9 promoter region was cloned and constructed in a vector of luciferase reporter gene. The 2.2-kb sequences were identified as having three candidate nuclear factor-κ B (NF-κB) binding sites: NF-κB I (-1418/-1409), NF-κB II (-626/-617), and NF-κB III (-353/-345). A series of reporter vectors with the mutated NF-κB sites of mmp-9 promoter sequences were constructed and transfected into H9c2 cells. The results show that the NF-κB II binding site (-626/-617) within the promoter region of mmp-9 plays a key role in upregulation of mmp-9 expression by TNF-α induction. In addition, we also first identified that the NF-κB I, similar to c-Rel, might be one of the NF-κB families to regulate mmp-9 expression.

Efficiency of DNA transfection of rat heart myoblast cells H9c2(2-1) by either polyethyleneimine or electroporation.

Expression of exogenous DNA in vitro is significantly affected by the particular transfection method utilized. In this study, we evaluated the efficiency of two transfection methods, chemically mediated polyethyleneimine (PEI) treatment and physically mediated electroporation, on a rat heart myoblast cell line, H9c2(2-1). After PEI transfection of pPgk-1/EGFP into H9c2(2-1) cells, EGFP expression could be easily detected by fluorospectrometer after 48 h (210 ± 12 RFU) and continued to increase after 72 h (243 ± 14 RFU). However, when H9c2(2-1) cells were transfected by electroporation (200 V, 500 µF, and one pulse), low level EGFP expression was observed after 48 h (49 ± 4 RFU) or 72 h (21 ± 14 RFU). In contrast, the easily transfectable control CHO-K1 cell line displayed a stronger EGFP expression than the H9c2(2-1) cells either by PEI or electroporation transfection. When transfection efficiencies were assayed by flow cytometry after 72 h, 13.6 ± 2.2% of PEI and 10.1 ± 1.5% of electroporation (250 V, 500 µF, and two pulses) transfected cells of H9c2(2-1) expressed EGFP, and PEI-transfected cells appeared to be less damaged (viability 93.6%) as compared to electroporation-transfected cells (39.5%). However, both PEI and electroporation (580 V, 50 Ω, and 50 µF) were effective for transfection of CHO-K1 with a higher efficiency, cell viability, and EGFP expression than H9c2(2-1). Our results indicate that the transfection efficiency of different methods varies among cell lines and that PEI is more efficient than electropolation for transfection of H9c2(2-1) whereas both PEI and electroporation are suitable for CHO-K1 transfection.

Potentiation of angiogenesis and regeneration by G-CSF after sciatic nerve crush injury.

Granulocyte colony-stimulating factor (G-CSF) demonstrates neuroprotective effects through different mechanisms, including mobilization of bone marrow cells. However, the influence of G-CSF-mediated mobilization of bone marrow-derived cells on injured sciatic nerves remains to be elucidated. The administration of G-CSF promoted a short-term functional recovery 7 days after crush injury in sciatic nerves. A double-immunofluorescence study using green fluorescent protein-chimeric mice revealed that bone marrow-derived CD34+ cells were predominantly mobilized and migrated into injured nerves after G-CSF treatment. G-CSF-mediated beneficial effects against sciatic nerve injury were associated with increased CD34+ cell deposition, vascular endothelial growth factor (VEGF) expression, and vascularization/angiogenesis as well as decreased CD68+ cell accumulation. However, cell differentiation and VEGF expression were not demonstrated in deposited cells. The results suggest that the promotion of short-term functional recovery in sciatic nerve crush injury by G-CSF involves a paracrine modulatory effect and a bone marrow-derived CD34+ cell mobilizing effect.

In vitro and ex vivo green fluorescent protein expression in alveolar mammary epithelial cells and mammary glands driven by the distal 5'-regulative sequence and intron 1 of the goat beta-casein gene.

The 5'-regulative sequence and intron 1 of the goat beta-casein gene from -4044 to +2123 bp was cloned and fused with the reporter gene of green fluorescent protein (GFP) to create a plasmid termed pGB562/GFP. To detect GFP expression, pGB562/GFP was transfected in vitro via liposomes into the mammary epithelial cell line NMuMG. Cells could not express GFP unless the transfected NMuMG cells lined up to create functional alveoli. These functional cells were cultured with lactogenic hormones, including insulin, dexamethasone and prolactin, and were grown on a layer of the extracellular matrix Matrigel. Green fluorescent protein expression levels in NMuMG cells were 25-, 55- and 42-fold those in the control group at 24, 48, and 72 h after pGB562/GFP transfection respectively. In addition, pGB562/GFP was transfected ex vivo by electroporation into mammary gland fragments and cells were then cultured in vitro with a supplement of lactogenic hormones. Strong GFP expression localized in fragments of the mammary gland was observed 24 h after gene transfer. The novel strategy of ex vivo gene transfer into mammary tissue using GFP as a reporter gene to detect the function of a tissue-specific promoter is efficient and convenient. The data obtained herein reveal that the 5'-regulative sequence and intron 1 of the 6.2 kb goat beta-casein gene can enhance the efficiency of transgene expression. Thus, the GB562 sequence may act as a good promoter and effectively elevate the production of exogenous protein in mammary glands.