Optimization of seeding density of OP9 cells to improve hematopoietic differentiation efficiency.

BACKGROUND: OP9 mouse stromal cell line has been widely used to induce differentiation of human embryonic stem cells (hESCs) into hematopoietic stem/progenitor cells (HSPCs). However, the whole co-culture procedure usually needs 14-18 days, including preparing OP9 cells at least 4 days. Therefore, the inefficient differentiation system is not appreciated. We aimed to optimize the culture conditions to improve differentiation efficiency. METHODS: In the experimental group, we set six different densities of OP9 cells and just cultured them for 24 h before co-culture, and in the control group, OP9 cells were cultured for 4 days to reach an overgrown state before co-culture. Then we compared the hematopoietic differentiation efficiency among them. RESULTS: OP9 cells were randomly assigned into two groups. In the experimental group, six different plated numbers of OP9 cells were cultured for 1 day before co-culture with hESCs. In contrast, in the control group, OP9 cells were cultured for 4 days at a total number of 3.1 × 104 cells/cm2 in a 6-well plate to reach an overgrown state before co-culture. Hematopoietic differentiation was evaluated with CD34 immunostaining, and compared between these two groups. We could not influence the differentiation efficiency of OP9 cells with a total number of 10.4 × 104 cells/cm2 in a 6-well plate which was cultured just for 1 day, followed by co-culture with hESCs. It reached the same differentiation efficiency 5 days earlier than the control group. CONCLUSION: The peak of CD34 + cells appeared 2 days earlier compared to the control group. A total number of 1.0 × 106 cells in a 6-well plate for OP9 cells was appropriate to have high differentiation efficiency.

[Mechanism of miR-155 Promoting Drug Resistance in Childhood Acute Lymphoblastic Leukemia by Regulating Wnt/ß-Catenin Signaling Pathway].

OBJECTIVE: To investigate the mechanism of miR-155 promoting drug resistance of children B-ALL to Ara-C by regulating Wnt/ß-Catenin signaling pathway. METHODS: The expression of miR-155 in bone marrow tissue and cell line of B-ALL was detected by PCR. The chemotherapy resistant strain REH/ Ara-C was constructed by using REH cells. REH/ Ara-C cells were transfected with miR-155 inhibitor. The proliferation of REH/Ara-C cells was detected by EdU. The apoptosis of REH/ Ara-C cells was detected by flow cytometry. The drug resistance of REH/Ara-C cells were analyzed by CCK-8 method and colony formation assay. The expression of Wnt/ß-Catenin signaling pathway related proteins were determined by Western blot. MiR-155 inhibitor and Wnt activator agonist were used to transfect REH/Ara-C cells, and their effects on cell proliferation, apoptosis and drug resistance were determined. RESULTS: Compared with normal tissues and cells, the expression level of miR-155 in B-ALL bone marrow tissue/cell line was increased (P<0.05); Compared with drug sensitive B-ALL tissues/cell lines, the expression level of miR-155 in drug resistant B-ALL tissues and cell lines was increased (P<0.05); Inhibition of miR-155 expression decreased the proliferation of REH/Ara-C cells (P<0.05), promoted apoptosis (P<0.05), enhanced the cytotoxicity of Ara-C (P<0.05), and inhibited Wnt/ß-Catenin signaling pathway related protein and MDR1 gene expression (P<0.05), which could be reversed by activating Wnt expression (P<0.05). CONCLUSION: The expression of miR-155 is up-regulated in bone marrow of children with B-ALL, which may be related to the activation of Wnt/ß-Catenin signaling pathway promotes the proliferation of B-ALL cells and inhibits apoptosis, which leads to chemotherapy resistance.

TRPV6 is a prognostic marker in early-stage cervical squamous cell carcinoma.

Transient receptor potential vanilloid 6 (TRPV6) has been shown to promote caner proliferation in several solid tumors, leading to unfavorable clinical outcomes. Our study aimed to elucidate the clinical significance of TRPV6 in patients with early-stage cervical squamous cell carcinoma (CSCC). The mRNA expression of TRPV6 was measured in 12 paired early-stage CSCC specimens and six cervical carcinoma cell lines using quantitative real-time PCR (qRT-PCR). Western blotting and immunohistochemistry (IHC) were employed to examine the protein expression level of TRPV6 in four paired specimens, 175 paraffin-embedded early-stage CSCC specimens, and 50 normal cervical tissues (NCTs), respectively. Statistical analyses were performed to evaluate the clinical significance of TRPV6 expression. The expressions of TRPV6 mRNA and protein were both significantly downregulated in early-stage CSCC tissues and cervical cancer cell lines. IHC analyses revealed that TRPV6 was downregulated in 136 (77.7 %) of 175 early-stage CSCC specimens. Moreover, TRPV6 expression in early-stage CSCC was significantly correlated with the tumor stage (P < 0.001), tumor growth type (P < 0.001), tumor size (P = 0.008), and differentiation grade (P = 0.003). The early-stage CSCC patients with a low TRPV6 expression level had a short progress-free survival (PFS) and overall survival (OS) duration. Univariate and multivariate analyses identified TRPV6 as an independent prognostic factor for early-stage CSCC patients' survival. We demonstrated that TRPV6 was downregulated in CSCC, which was correlated with unfavorable survival outcomes of early-stage CSCC patients. TRPV6 may be used as a novel prognostic marker for early-stage CSCC.