Lipofection of Single Guide RNA Targeting MMP8 Decreases Proliferation and Migration in Lung Adenocarcinoma Cells.

Background and Objectives: Matrix metalloproteinases (MMP) have been implicated as major determinants of tumour growth and metastasis, which are considered two of the main hallmarks of cancer. The interaction of MMP8 and other signalling molecules within and adjacent tumoral tissues, including immune cells, are rather elusive, particularly of adenocarcinoma cell type. In this study, we aimed to investigate the role of MMP8 in non-small cell lung cancer proliferation and invasiveness potential. Materials and Methods: We individually lipofected with two different single guide RNA (sgRNAs) that specifically targeted on MMP8, with CRISPR-Cas 9 protein into the cells. Results: Our results clearly indicated that the lipofection of these complexes could lead to reduced ability of A549 cells to survive and proliferate to form colonies. In addition, when compared to non-transfected cells, the experimental cell groups receiving sgRNAs demonstrated relatively decreased migration rate, hence, wider wound gaps in scratch assay. The quantitative real time-polymerase chain reaction (qRT-PCR) demonstrated significant reduction in the MAP-K, survivin and PI3-K gene expression. MMP8 might have protective roles over tumour growth and spread in our body. Conclusions: The delivery of sgRNAs targeting on the MMP8 gene could induce tumour cell death and arrest cell migratory activity.

Intrastriatal transplantation of stem cells from human exfoliated deciduous teeth reduces motor defects in Parkinsonian rats.

BACKGROUND: This study explored the neural differentiation and therapeutic effects of stem cells from human exfoliated deciduous teeth (SHED) in a rat model of Parkinson's disease (PD). METHODS: The SHED were isolated from fresh dental pulp and were induced to differentiate to neurons and dopamine neurons by inhibiting similar mothers against dpp (SMAD) signaling with Noggin and increase conversion of dopamine neurons from SHED with CHIR99021, Sonic Hedgehog (SHH) and FGF8 in vitro. The neural-primed SHED were transplanted to the striatum of 6-hydroxydopamine (6-OHDA)-induced PD rats to evaluate their neural differentiation and functions in vivo. RESULTS: These SHED were efficiently differentiated to neurons (62.7%) and dopamine neurons (42.3%) through a newly developed method. After transplantation, the neural-induced SHED significantly improved recovery of the motor deficits of the PD rats. The grafted SHED were differentiated into neurons (61%), including dopamine neurons (22.3%), and integrated into the host rat brain by forming synaptic connections. Patch clamp analysis showed that neurons derived from grafted SHED have the same membrane potential profile as dopamine neurons, indicating these cells are dopamine neuron-like cells. The potential molecular mechanism of SHED transplantation in alleviating motor deficits of the rats is likely to be mediated by neuronal replacement and immune-modulation as we detected the transplanted dopamine neurons and released immune cytokines from SHED. CONCLUSION: Using neural-primed SHED to treat PD showed significant restorations of motor deficits in 6-OHDA-induced rats. These observations provide further evidence that SHED can be used for cell-based therapy of PD.

Effect of 660-nm LED photobiomodulation on the proliferation and chondrogenesis of meniscus-derived stem cells (MeSCs).

Meniscus-derived stem cells (MeSCs), a unique type of MSC, have outstanding advantages in meniscal cytotherapy and tissue engineering, but the effects and molecular mechanisms of PBM on MeSCs are still unclear. We used 660-nm LED light with different energy densities to irradiate six human MeSC samples and tested their proliferation rate via cell counting, chondrogenic differentiation capacity via the DMMB assay, mitochondrial activity via the MTT assay, and gene expression via qPCR. The proliferation ability, chondrogenic capacity and mitochondrial activity of the 18 J/cm2 group were greater than those of the 4 J/cm2 and control groups. The mRNA expression levels of Akt, PI3K, TGF-ß3, Ki67 and Notch-1 in the 18 J/cm2 group were greater than those in the other groups in most samples. After chondrogenic induction, the expression of Col2A1, Sox9 and Aggrecan in the 18 J/cm2 group was significantly greater than that in the 4 J/cm2 and control groups in most of the samples. The variation in the MTT values and Src, PI3K, Akt, mTOR and GSK3ß levels decreased with time. The results showed that 660-nm LED red light promoted proliferation and chondrogenic differentiation and affected the gene expression of MeSCs, and the effects on gene expression and mitochondrial activity decreased with time.

Epidemiology of human respiratory viruses in children with acute respiratory tract infections in Jinan, China.

The viral etiologies of UTRIs and LTRIs in children in Jinan city were investigated between July 2009 and June 2010. Nasal and throat swabs were collected from 397 children with URTIs and bronchoalveolar lavage fluid specimens were collected from 323 children with LRTIs. RT-PCR/PCR was used to examine all samples for IFV, PIV, RSV, RV, hMPV, HBoV, CoV, ADV, RSV, and EV. Viral pathogens were detected in 47.10% of URTI samples and 66.57% samples, and the incidence of viral coinfection was 5.29% and 21.05%, respectively. IFV was the most common virus in URTIs, with a detection rate of 19.40%, followed by PIV (10.83%), RV (10.58%), and EV (6.30%). For LRTIs, PIV and RV were both detected in 27% of samples, followed by RSV (9.91%), HBoV (8.36%), IFV (5.57%), and hMPV (5.57%). RSV and HBoV were more prevalent in the youngest children of no more than six months. Meanwhile, RV, PIV, and RSV were the most frequent viruses combined with bacterial pathogens in LRTIs. In conclusion, the spectrum of respiratory virus infections in URTIs and LRTIs differed in terms of the most common pathogens, seasonal distribution, and coinfection rate.

Viral aetiology in adults with acute upper respiratory tract infection in Jinan, Northern China.

Our study investigated the epidemiology of respiratory viruses in adult patients with upper respiratory tract infection (URTI) between August 2009 and September 2010 in Jinan, northern China. Nasal and throat swabs (n = 596) were collected from adult patients with URTIs. Nine respiratory-related viruses, including IFV, PIV, HRV, HMPV, HBoV, HCoV, ADV, RSV, and EV, were detected in all samples by conventional and reverse transcription polymerase chain reactions. Positive detection rate for respiratory virus was 38.76% and codetection rate was 4.70% in adults with acute respiratory tract infections. IFV (20.81%) was the dominant agent detected and IFVB had a higher incidence (12.58%) than IFVA (7.72%). Detection rates of 8.22%, 5.03%, 3.69%, and 2.52% were observed for HBoV, HRV, EV, and RSV, respectively. HCoV had the lowest detection rate of 0.50%. HBoV, HRV, EV, and ADV infection rates were higher in the 14-25-year-old group than in the 26-65-year-old group. Codetection rates were higher (7.52%) in the 14-25-year-old group than in the older age group (2.64%). The spectrum of respiratory virus infection in adult patients with URTIs was different in Jinan compared with other cities in China.

Case Report: Cord blood-derived natural killer cells as new potential immunotherapy drug for solid tumor: a case study for endometrial cancer.

Adoptive transfer of natural killer (NK) cells represents a viable treatment method for patients with advanced malignancies. Our team previously developed a simple, safe, and cost-effective method for obtaining high yields of pure and functional NK cells from cord blood (CB) without the need for cell sorting, feeder cells, or multiple cytokines. We present the case of a 52-year-old female patient diagnosed with poorly differentiated stage IVB (T3N2M1) endometrial cancer, who exhibited leukemoid reaction and pretreatment thrombocytosis as paraneoplastic syndromes. The patient received two courses of CB-derived NK (CB-NK) cell immunotherapy between March and September 2022, due to her extremely low NK cell activity. Two available CB units matched at 8/10 HLA with KIR-mismatch were chosen, and we were able to produce NK cells with high yield (>1.0×1010 NK cells), purity (>90%), and function (>80%) from CB without cell sorting, feeder cells, or multiple cytokines. These cells were then adoptively transferred to the patient. No adverse effects or graft-versus-host disease were observed after infusion of CB-NK cells. Our clinical experience supports the efficacy of CB-NK cell treatment in increasing NK cell activity, depleting tumor activity, improving quality of life, and reducing the size of abdominal and pelvic masses with the disappearance of multiple lymph node metastases through the regulation of systemic antitumor immunity. Remarkably, the white blood cell and platelet counts decreased to normal levels after CB-NK cell immunotherapy. This clinical work suggests that CB-NK cell immunotherapy holds promise as a therapeutic approach for endometrial cancer.

Establishment and characterization of human induced pluripotent stem cell line from a Parkinson's disease patient harboring VPS13A gene mutation.

Mutations in VPS13 gene have been recently reported as a genetic cause of Parkinson's disease (PD). In this study, we isolated the skin fibroblasts from a PD patient harboring VPS13A gene mutation (c. 4282_4289delinsA) and reprogrammed the fibroblasts to a novel patient-specific induced pluripotent stem cell (iPSC) line LCPHi002-A using transgene-free episomal plasmids to express OCT3/4, SOX2, KLF4, L-MYC, and LIN28. The LCPHi002-A line showed the normal karyotype, expression of pluripotency markers, and had multi-lineage differentiation capacity in vivo. This iPSC line of LCPHi002-A could be used for studying pathogenic mechanisms of PD.

Lung development, repair and cancer: A study on the role of MMP20 gene in adenocarcinoma.

Multiple matrix metalloproteinases have significant roles in tissue organization during lung development, and repair. Imbalance of proteinases may lead to chronic inflammation, changes in tissue structure, and are also highly associated to cancer development. The role of MMP20 is not well studied in lung organogenesis, however, it was previously shown to be present at high level in lung adenocarcinoma. The current study aimed to identify the functional properties of MMP20 on cell proliferation and motility in a lung adenocarcinoma in vitro cell model, and relate the interaction of MMP20 with other molecular signalling pathways in the lung cells after gaining tumoral properties. In this study, two different single guide RNA (sgRNAs) that specifically targeted on MMP20 sites were transfected into human lung adenocarcinoma A549 cells by using CRISPR-Cas method. Following that, the changes of PI3-K, survivin, and MAP-K mRNA gene expression were determined by Real-Time Polymerase Chain Reaction (RT-PCR). The occurrence of cell death was also examined by Acridine Orange/Propidium Iodide double staining. Meanwhile, the motility of the transfected cells was evaluated by wound healing assay. All the data were compared with non-transfected cells as a control group. Our results demonstrated that the transfection of the individual sgRNAs significantly disrupted the proliferation of the A549 cell line through suppression in the gene expression of PI3-K, survivin, and MAP-K. When compared to non-transfected cells, both experimental cell groups showed reduction in the migration rate, as reflected by the wider gaps in the wound healing assay. The current study provided preliminary evidence that MMP20 could have regulatory role on stemness and proliferative genes in the lung tissues and affect the cell motility. It also supports the notion that targeting MMP20 could be a potential treatment mode for halting cancer progression.

Hematopoietic stem and progenitor cells can be enriched by implanting biomaterial into spatium intermusculare.

Hematopoietic stem and progenitor cells (HSPCs) have been used successfully to treat patients with cancer and disorders of the blood and immune systems. In this study, we tried to enrich HSPCs by implanting biomaterials into the spatium intermusculare of mice hind limbs. Gelatine sponges were implanted into the spatium intermusculare of mice and then retrieved after 12 days. The presence of HSPCs in the migrating cells (MCs) was detected by phenotypically probing with CD34(+)Sca-1(+) and functionally confirming the presence of using colony-forming cell assay and assessing the long-term reconstitution ability. The frequency of CD34(+), Sca-1(+), and CD34(+)Sca-1(+) cells and colony formation unit in the MCs was much higher than that in the bone marrow (BM). Moreover, transplanted MCs were able to home to BM, muscle, and spleen, which induced an efficient long-term hematopoietic reconstitution in vivo. In addition, HSPCs within the MCs originated from the BM. Furthermore, the administration of G-CSF greatly reduced the time of implantation, and increased the number of MCs and frequency of HSPCs in the MCs. These data provide compelling evidence that HSPCs can be enriched by implanting biomaterial into spatium intermusculare. Implantation of biomaterial may be seen as the first step to a proof of their applicability to clinical practice in enriching HSPCs.

Comparative analysis of human mesenchymal stem cells from bone marrow and adipose tissue under xeno-free conditions for cell therapy.

INTRODUCTION: Mesenchymal stem cells (MSCs) are promising candidates for cell-based therapies. Human platelet lysate represents an efficient alternative to fetal bovine serum for clinical-scale expansion of MSCs. Different media used in culture processes should maintain the biological characteristics of MSCs during multiple passages. However, bone marrow-derived MSCs and adipose tissue-derived MSCs have not yet been directly compared with each other under human platelet lysate conditions. This study aims to conduct a direct head-to-head comparison of the biological characteristics of the two types of MSCs under human platelet lysate-supplemented culture conditions for their ability to be used in regenerative medicine applications. METHODS: The bone marrow- and adipose tissue-derived MSCs were cultured under human platelet lysate conditions and their biological characteristics evaluated for cell therapy (morphology, immunophenotype, colony-forming unit-fibroblast efficiency, proliferation capacity, potential for mesodermal differentiation, secreted proteins, and immunomodulatory effects). RESULTS: Under human platelet lysate-supplemented culture conditions, bone marrow- and adipose tissue-derived MSCs exhibited similar fibroblast-like morphology and expression patterns of surface markers. Adipose tissue-derived MSCs had greater proliferative potential than bone marrow-derived MSCs, while no significantly difference in colony efficiency were observed between the two types of cells. However, bone marrow-derived MSCs possessed higher capacity toward osteogenic and chondrogenic differentiation compared with adipose tissue-derived MSCs, while similar adipogenic differentiation potential wase observed between the two types of cells. There were some differences between bone marrow- and adipose tissue-derived MSCs for several secreted proteins, such as cytokine (interferon-γ), growth factors (basic fibroblast growth factor, hepatocyte growth factor, and insulin-like growth factor-1), and chemokine (stem cell-derived factor-1). Adipose tissue-derived MSCs had more potent immunomodulatory effects than bone marrow-derived MSCs. CONCLUSIONS: Adipose tissue-derived MSCs have biological advantages in the proliferative capacity, secreted proteins (basic fibroblast growth factor, interferon-γ, and insulin-like growth factor-1), and immunomodulatory effects, but bone marrow-derived MSCs have advantages in osteogenic and chondrogenic differentiation potential and secreted proteins (stem cell-derived factor-1 and hepatocyte growth factor); these biological advantages should be considered systematically when choosing the MSC source for specific clinical application.