Guanghui Cui

Mesenchymal stem cells derived from different origins have unique sensitivities to different chemotherapeutic agents

BMSCs (bone‐marrow‐derived mesenchymal stem cells) and ADSCs (adipose tissue‐derived mesenchymal stem cells) are virtually identical in cell surface marker profile and differentiation potential. These cell populations have promising characteristics for clinical application. We have investigated the sensitivity of these cell populations to various chemotherapeutic agents by testing the inhibition of cell proliferation, low molecular DNA bands formation, in situ apoptosis, apoptosis‐related gene expression and cell senescence after treatment. BU (busulfan), methotrexate and doxorubicin treatment led to a marked and dose‐dependent reduction in cell viability compared with 5‐FU (5‐fluorouracil) treatment. Different expression patterns of apoptosis‐related genes were found in the BMSCs and ADSCs following treatment with the agents, but no low molecular mass DNA bands were detected. BMSCs had a higher percentage of apoptotic and senescent cells following treatment with chemotherapeutic agents compared with ADSCs. These findings suggest that these two cell populations respond differently to chemotherapy treatment. ADSCs are more resistant than BMSCs to chemotherapy‐induced senescence and apoptosis, indicating that they might be more advantageous to use in the clinic than BMSCs.

Cluster characterization of mouse embryonic stem cell-derived pluripotent embryoid bodies in four distinct developmental stages

The formation of embryoid bodies (EBs) is the principal step in the differentiation of embryonic stem (ES) cells. In this study, the morphological characteristics and gene expression patterns of EBs related to the sequential stages of embryonic development were well defined in four distinct developmental groups over 112 days of culture: early-stage EBs groups (1-7 days of differentiation), mid-stage EBs groups (9-15 days of differentiation), maturing EBs groups (17-45 days of differentiation) and matured EBs groups (50 days of differentiation). We first determined definite histological location of apoptosis within EBs and the sequential expression of molecular markers representing stem cells (Oct4, SSEA-1, Sox-2 and AKP), germ cells (Fragilis, Dazl, c-kit, StellaR, Mvh and Stra8), ectoderm (Neurod, Nestin and Neurofilament), mesoderm (Gata-1, Flk-1 and Hbb) and endoderm (AFP and Transthyretin). Our results revealed that developing EBs possess either pluripotent stem cell or germ cell states and that three-dimensional aggregates of EBs initiate mES cell differentiation during prolonged culture in vitro. Therefore, we suggest that this EB system to some extent recapitulates the early developmental processes occurring in vivo.

Gossypol repressed the gap junctional intercellular communication between Sertoli cells by decreasing the expression of Connexin43.

Previous studies showed that gossypol could block the gap junctional intercellular communication (GJIC) between cultured cells. The present study was designed to investigate the effects of gossypol on the GJIC and the expression of connexin43 (Cx43) in the cultured cells. A Sertoli cell line, TM4, was treated with different concentrations of gossypol 1.25, 2.5, 5, and 10micromol/L for 6, 12, 24, and 48h. Cell viability was assessed with CCK-8 assay. GJIC in the cells was determined using the scrape loading and dye transfer (SLDT) assay; the expression of Cx43 was detected by RT-PCR, immunofluorescence and Western blot analysis. The SLDT assay showed gossypol significantly decreased GJIC between adjacent cells. RT-PCR, immunofluorescence and Western blot analyses demonstrated the expression of Cx43 in TM4 cells. The expression of Cx43 was gradually decreased with the increasing concentrations of gossypol, and the effect occurred as early as 6h after the treatment and continued until 48h. These results suggested that gossypol impaired GJIC by decrease of Cx43 expression in the cells, which is important for Sertoli cells to regulate spermatogenesis.

Tumor suppressive microRNA-200a inhibits renal cell carcinoma development by directly targeting TGFB2

A large body of evidence indicates that microRNAs play a critical role in tumor initiation and progression by negatively regulating oncogenes or tumor suppressor genes. Here, we report that the expression of miR-200a was notably downregulated in 45 renal cell carcinoma (RCC) samples. Restoration of miR-200a suppressed cell proliferation, migration, and invasion in two RCC cell lines. Furthermore, we used an epithelial-to-mesenchymal transition PCR array to explore the putative target genes of miR-200a. By performing quantitative real-time PCR, ELISA, and luciferase reporter assays, transforming growth factor beta2 (TGFB2) was validated as a direct target gene of miR-200a. Moreover, siRNA-mediated knockdown of TGFB2 partially phenocopied the effect of miR-200a overexpression. These results suggest that miR-200a suppresses RCC development via directly targeting TGFB2, indicating that miR-200a may present a novel target for diagnostic and therapeutic strategies in RCC.

Overexpression of Cyclooxygenase-1 Correlates with Poor Prognosis in Renal Cell Carcinoma

The aim of this study was to evaluate expression of COX-1 in renal cell carcinoma (RCC) and its prognostic value. mRNA of COX-1 was detected in 42 paired RCC and adjacent normal tissues with quantitative real- time polymerase chain reaction (qRT-PCR). Expression of COX-1 was also evaluated in 196 RCC sections and 91 adjacent normal tissues with immunohistochemistry. Statistical analysis was performed to assess COX-1 expression in RCC and its prognostic significance. The results of qRT-PCR showed mRNA levels of COX-1 in RCC tissues to be significantly higher than that in adjacent normal tissues (p < 0.001). Immunohistochemical assays also revealed COX-1 to be overexpressed in RCC tissues (p < 0.001). Statistical analysis demonstrated high expression of COX-1 was correlated with tumour size (p = 0.002), pathological stage (p = 0.003), TNM stage (p = 0.003, 0.007, 0.027, respectively), and tumour recurrence (p < 0.001). Survival analysis indicated patients with high expression of COX-1 had shorter survival time (p < 0.001), and COX-1 was an independent predictor. This is the first study to reveal overexpression of COX-1 in RRC and point to use as a prognostic marker in affected patients.

Rat bone marrow derived mesenchymal progenitor cells support mouse ES cell growth and germ‐like cell differentiation

Mouse embryonic fibroblasts (MEFs) have been used as feeder cells to support the growth of mouse embryonic stem cell (mESC) and primordial germ cells (PGC) in culture for many years. However, MEF preparation is a complex and tedious task. Recently, there are reports indicating that the microenvironment provided by bone marrow stromal cells could support the survival of embryonic‐like stem cells in bone marrow. In this report, rat bone marrow derived mesenchymal progenitor cells (MPC) were used as feeder cells to culture mouse Oct4‐GFP ES cell and ES cell derived germ cells. FACS results show that similar to MEF, rat MPC could efficiently support growth of the mouse Oct4‐GFP ES cell line in culture (MPC 85.5 ± 5.1% vs MEF 84.1 ± 6.2%). ES cells could be subcultured for >15 passages without losing morphological characteristics. The cultured cells expressed stem cell marker alkaline phosphatase, Oct4, Sox2, and SSEA‐1. Furthermore, rat MPC cells were able to support survival of germ cells isolated from mouse Oct4‐GFP ES cell formed embryoid bodies (EB). After induction by retinoic acid for 7 days, some isolated cells differentiated to spermatogonial stem‐like cells, expressing Mvh, Stra‐8, Hsp90‐α, integrinβ1 and α6. Compared with traditional MEF culture systems, the rat MPC culture system is effective in supporting ES cell growth and is easy to prepare.

LIN28A inhibits lysosome‑associated membrane glycoprotein 1 protein expression in embryonic stem and bladder cancer cells

Tumor cells and embryonic stem cells (ESCs) have similar transcription mechanisms. LIN28A is an important factor in tumor cells and ESCs, it is an inhibitor of intracellular endoplasmic reticulum (ER)‑related protein translation in ESCs. The present study aimed to examine the effects of LIN28A on an ER‑related protein, lysosome‑associated membrane glycoprotein 1 (LAMP1), in human bladder cancer cells and mouse (m)ESCs, using reverse transcription‑quantitative polymerase chain reaction and western blotting to detect the expression of LAMP1 mRNA and protein, respectively, following LIN28A knockdown. LIN28A was revealed to promote the proliferation, migration and invasion in human bladder cancer cells. These data suggested similarities between ESC cells and cancer cells and may provide novel ideas for the use of induced embryonic stem cell differentiation to treat tumors.

Induction of dedifferentiated male mouse adipose stromal vascular fraction cells to primordial germ cell-like cells

The adipose stromal vascular fraction (SVF) contains abundant mesenchymal stem cell populations that have a limited ability to self‐renew and differentiate. Male mouse adipose SVF cells were dedifferentiated by reprogramming factors (c‐Myc, Oct4, Sox2, and Klf4) to form embryonic stem cell‐like cells (ESCLCs), which upgraded their limited differentiation potential. The ESCLCs were induced to differentiate toward epiblast‐like cells (EpiLCs) and primordial germ cell‐like cells (PGCLCs) by culturing in media supplied with activin A and BMP‐4, respectively. The derived ESCLCs possess embryonic stem cell features and can automatically form embryonic bodies. After culture in EpiLC induction medium for 2–3 days, ESCLCs formed flattened epithelial structures that were different from their original water drop‐like colonies, and the expression of pluripotency‐related genes decreased. When the cells that had been cultured in EpiLC induction medium for 2 days were isolated and cultured in PGCLC induction medium for 4–6 days, they formed typical water drop‐like colonies again. Moreover, expression of the pluripotency‐related genes and the primordial germ cell (PGC) specification‐related genes increased. During progression from ESCLCs toward EpiLCs and PGCLCs, the levels of histone methylases H3K9me2 and H3K27me3 kept changing, which resembled those seen in PGC specification. The derived PGCLCs expressed SSEA‐1, Blimp‐1, and Stella. Furthermore, methylation of Igf2r and Snrpn was retained, but H19 and Kcnq1ot1 methylation levels were slightly reduced compared to non‐PGCLCs, suggesting that the derived PGCLCs may have initiated the process of imprint erasure.

Growth and differentiation of embryoid bodies derived from nouse embryonic stem cells

Differentiation of Embryonic stem (ES) cells generally occurs after formation of three-dimensional cell aggregates, known as embryoid bodies (EBs). Even though the formation of an EB in a suspension culture has been adopted as an initial strategy for the ES cells differentiation, not much is known about the characteristics of EB cells. To follow differentiation of EBs in vitro, the process of EB formation and genes that were transiently expressed at different stages at 1, 3, 5, 7, 9, 11, 13, 15, 21, 33 and 57-day old EBs were determined. The results showed that various types of differentiated cells, such as neural cells, cardiomyocytes, skeletal muscle cells and adipocytes were found in the EBs and identified. EBs expressed embryonic specific genes in a stage specific manner, Nestin and Neurod were consider as ectoderm marker, Flk-1 and Desmin as mesoderm marker, GATA-4 and AFP as endoderm marker, which were either up-regulated gradually or immediately, or begin to down-regulate in maturing or fully matured EBs. A more refined analysis showed the EBs contained germ like cells, specific markers as Oct-3/4, c-kit, Mvh, DAZL, Stra8 were expressed during the process of EBs in vitro differentiation. In conclusion, our results demonstrated the potential of mouse ES cells to differentiate into all three germ layers cells and express proper markers of EBs, and also revealed that EBs can differentiate into fully differentiated cells and form primitive tissues as ovarian-like structure, oocyte-specific markers such as Fig- and ZP3 were detected. This procedure will helpful for the human embryonic development research and discrete lineage for the cell-based replacement therapy.