Colony-forming Efficiency Research Articles

JF-305, a pancreatic cancer cell line is highly sensitive to the PARP inhibitor olaparib.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a DNA nick sensor involved in the base excision repair (BER) pathway. Olaparib, a PARP inhibitor, has demonstrated antitumor activity in homologous recombination (HR)-deficient cancers. To extend this specific therapy to other types of carcinomas, a panel of 11 different cancer cells were screened in the present study. JF-305, a pancreatic cancer cell line of Chinese origin, demonstrated sensitivity to the PARP inhibitor 6(5H)-phenanthridinone. In the present study, 3 μM olaparib conferred a cell survival rate of 25% following four days of treatment. The colony formation efficiency was 83% at 10 nM, and dropped to 12% at 1 μM following seven days of treatment. Furthermore, olaparib induced cell cycle arrest in the S and G2/M phases prior to the initiation of apoptosis. Although the incidence of double-strand breaks (DSBs) was increased in the olaparib-treated JF-305 cells, the RAD51 foci were well formed at the sites of γ-H2AX recruitment, indicating an activated HR mechanism. Furthermore, tumor growth was reduced by 49.8% following 22 days of consecutive administration of 10 mg/kg olaparib in the JF-305 xenograft mouse model. In summary, the JF-305 cell line was sensitive to olaparib and provided a prospective model for the preclinical assessment of PARP inhibitors in the therapy of pancreatic cancer.

The effects of spheroid formation of adipose-derived stem cells in a microgravity bioreactor on stemness properties and therapeutic potential

Adipose-derived stem cells (ADSCs) represent a valuable source of stem cells for regenerative medicine, but the loss of their stemness during in vitro expansion remains a major roadblock. We employed a microgravity bioreactor (MB) to develop a method for biomaterial-free-mediated spheroid formation to maintain the stemness properties of ADSCs. ADSCs spontaneously formed three-dimensional spheroids in the MB. Compared with monolayer culture, the expression levels of E-cadherin and pluripotent markers were significantly upregulated in ADSC spheroids. Spheroid-derived ADSCs exhibited increased proliferative ability and colony-forming efficiency. By culturing the spheroid-derived ADSCs in an appropriate induction medium, we found that the multipotency differentiation capacities of ADSCs were significantly improved by spheroid culture in the MB. Furthermore, when ADSCs were administered to mice with carbon tetrachloride-induced acute liver failure, spheroid-derived ADSCs showed more effective potentials to rescue liver failure than ADSCs derived from constant monolayer culture. Our results suggest that spheroid formation of ADSCs in an MB enhances their stemness properties and increases their therapeutic potential. Therefore, spheroid culture in an MB can be an efficient method to maintain stemness properties, without the involvement of any biomaterials for clinical applications of in vitro cultured ADSCs.

BMP-2 inhibits tumor-initiating ability in human renal cancer stem cells and induces bone formation.

We have previously shown that BMP-2 induces bone formation and inhibits tumorigenicity of cancer stem cells (CSCs) in a human osteosarcoma OS99-1 cell line. In this study, we sought to determine whether BMP-2 can similarly induce bone formation and inhibit the tumorigenicity of renal CSCs identified based on aldehyde dehydrogenase (ALDH) activity in renal cell carcinoma (RCC) cell lines and primary tumors. Using a xenograft model in which cells from human RCC cell lines ACHN, Caki-2, and primary tumors were grown in NOD/SCID mice, renal CSCs were identified as a subset of ALDH(br) cells. The ALDH(br) cells possessed a greater colony-forming efficiency, higher proliferative output, increased expression of stem cell marker genes Oct3/4A, Nanog, renal embryonic marker Pax-2, and greater tumorigenicity compared to cells with low ALDH activity (ALDH(lo) cells), generating new tumors with as few as 25 cells in mice. In vitro, BMP-2 was found to inhibit the ALDH(br) cell growth, down-regulate the expression of embryonic stem cell markers, and up-regulate the transcription of osteogenic markers. In vivo, all animals receiving a low number of ALDH(br) cells (5 × 10(3)) from ACHN, Caki-2, and primary tumor xenografts treated with 30 µg BMP-2 per animal showed limited tumor growth with significant bone formation, while untreated cells developed large tumor masses without bone formation. These results suggest that BMP-2 inhibits the tumor-initiating ability of renal CSCs and induces osseous bone formation. BMP-2 may therefore provide a beneficial strategy for human RCC treatment by targeting the CSC-enriched population.

Reconstruction of Highly Proliferative Auto-Tissue-Engineered Lamellar Cornea Enhanced by Embryonic Stem Cell.

To increase the epithelial proliferation of an auto-tissue-engineered lamellar cornea, 3.0 × 10(6) corneal epithelial cells (CECs) were combined with 3 × 10(5) mouse embryonic stem cells (ESCs) pretransfected with the HSV-tk gene (CECs + ESCs-TK group), and 3.3×10(6) corneal epithelial cells (CECs group) were seeded between the acellular porcine corneal stroma and the amniotic membrane using the centrifugal cell seeding method. After 4 days of perfusion culture (treatment with ganciclovir starting on day 2), a thicker corneal epithelium (four to five layers) formed in the CECs + ESCs-TK group compared with that observed in the CECs group (two to three layers). More stem/progenitor cell (K3 -, p63+, ABCG2+, and integrin-β1+) and proliferation phenotypes (Ki67+) were measured in the CECs + ESCs-TK group compared with the CECs group using immunofluorescence staining, real-time quantitative reverse transcription polymerase chain reaction, and flow cytometry. Consistent with these findings, the colony-forming efficiency and cellular doubling time were significantly different between the CECs + ESCs-TK group (16.18% ± 3.98%, 28.45 ± 2.03 h) and CECs group (11.96% ± 2.60%, 36.3 ± 1.15 h). In a rabbit lamellar transplantation model, the CECs + ESCs-TK group had better epithelial barrier functions and wound healing abilities compared with the CECs group. Furthermore, ESCs-TK could be completely and safely removed by ganciclovir. Thus, the ESCS-TK coculture system could serve as a potential strategy for corneal tissue engineering.

Magnetically labeled feeder system for mouse pluripotent stem cell culture

We report here a magnetically labeled feeder system for mouse embryonic stem/induced pluripotent stem (ES/iPS) cells. Magnetic attraction of feeder cells labeled with magnetite nanoparticles significantly increased ES/iPS colony-forming efficiency. Magnetic labeling of feeder cells also facilitated separation of ES/iPS cells from feeder cells.

Characterization of biomaterial-free cell sheets cultured from human oral mucosal epithelial cells.

The purpose of this study was to report the characteristics of biomaterial-free sheets cultured from human oral mucosal epithelial cells without fibrin support, in vitro and after transplantation to limbal-deficient models. Human oral mucosal epithelial cells and limbal epithelial cells were cultured for 2 weeks, and the colony-forming efficiency (CFE) rates were compared. Markers of stem cells (p63), cell proliferation (Ki-67) and epithelial differentiation (cytokeratin; K1, K3, K4, K13) were observed in colonies and in biomaterial-free sheets. Biomaterial-free sheets which had been detached with 1% dispase or biomaterial-free sheets generated by fibrin support were transplanted to 12 limbal-deficient rabbit models. In vitro cell viability, in vivo stability and cytokeratin characteristics of biomaterial-free sheets were compared with those of sheets formed by fibrin-coated culture 1 week after transplantation. Mean CFE rate was significantly higher in human oral mucosal epithelial cells (44.8%) than in human limbal epithelial cells(17.7%). K3 and K4 were well expressed in both colonies and sheets. Biomaterial-free sheets had two to six layers of stratified cells and showed an average of 79.8% viable cells in the sheets after detachment. Cytokeratin expressions of biomaterial-free sheets were comparable to those of sheets cultured by fibrin support, in limbal-deficient models. Both p63 and Ki-67 were well expressed in colonies, isolated sheets and sheets transplanted to limbal-deficient models. Our results suggest that biomaterial-free sheets cultured from human oral mucosal epithelial cells without fibrin support can be an alternative option for cell therapy in use for the treatment of limbal-deficient diseases. Copyright © 2014 John Wiley & Sons, Ltd.

Effects of melatonin on the proliferation and apoptosis of sheep granulosa cells under thermal stress.

The cross-talk between oocyte and somatic cells plays a crucial role in the regulation of follicular development and oocyte maturation. As a result, granulosa cell apoptosis causes follicular atresia. In this study, sheep granulosa cells were cultured under thermal stress to induce apoptosis, and melatonin (MT) was examined to evaluate its potential effects on heat-induced granulosa cell injury. The results demonstrated that the Colony Forming Efficiency (CFE) of granulosa cells was significantly decreased (heat 19.70% ± 1.29% vs. control 26.96% ± 1.81%, p < 0.05) and the apoptosis rate was significantly increased (heat 56.16% ± 13.95%vs. control 22.80% ± 12.16%, p < 0.05) in granulosa cells with thermal stress compared with the control group. Melatonin (10−7 M) remarkably reduced the negative effects caused by thermal stress in the granulosa cells. This reduction was indicated by the improved CFE and decreased apoptotic rate of these cells. The beneficial effects of melatonin on thermal stressed granulosa cells were not inhibited by its membrane receptor antagonist luzindole. A mechanistic exploration indicated that melatonin (10−7 M) down-regulated p53 and up-regulated Bcl-2 and LHR gene expression of granulosa cells under thermal stress. This study provides evidence for the molecular mechanisms of the protective effects of melatonin on granulosa cells during thermal stress.

Biphenyl-4-carboxylic acid [2-(1H-indol-3-yl)-ethyl]-methylamide (CA224), a nonplanar analogue of fascaplysin, inhibits Cdk4 and tubulin polymerization: evaluation of in vitro and in vivo anticancer activity.

Biphenyl-4-carboxylic acid-[2-(1H-indol-3-yl)-ethyl]-methylamide 1 (CA224) is a nonplanar analogue of fascaplysin (2) that specifically inhibits Cdk4-cyclin D1 in vitro. Compound 1 blocks the growth of cancer cells at G0/G1 phase of the cell cycle. It also blocks the cell cycle at G2/M phase, which is explained by the fact that it inhibits tubulin polymerization. Additionally, it acts as an enhancer of depolymerization for taxol-stabilized tubulin. Western blot analyses of p53-positive cancer cells treated with compound 1 indicated upregulation of p53, p21, and p27 proteins together with downregulation of cyclin B1 and Cdk1. Compound 1 selectively induces apoptosis of SV40 large T-antigen transformed cells and significantly reduces colony formation efficiency, in a dose-dependent manner, of lung cancer cells. It is efficacious at 1/10th of the MTD against human tumors derived from HCT-116 and NCI-H460 cells in SCID mouse models. The promising efficacy of compound 1 in human xenograft models as well as its excellent therapeutic window indicates its potential for clinical development.

G-patch domain containing 2, a gene highly expressed in testes, inhibits nuclear factor-κB and cell proliferation.

G-patch domain containing 2 (GPATC2), a human gene that is highly expressed in the testes, was implicated as a novel cancer/testis antigen. The present study investigated GPATC2 expression in a number of human cell lines and rat tissues, and its potential biological function in 293T cells. Semi‑quantitative reverse transcription-polymerase chain reaction analysis showed that GPATC2 was widely expressed in 15 human cell lines (representing different lineages) and in 11 different rat tissues, and that the GPATC2 mRNA relative expression level was significantly higher in the testis than it was in other tissues. 293T cells were transiently transfected with GPATC2-p enhanced green fluorescent protein (EGFP)‑N1 or GPATC2-pEGFP-C3 and the nuclei were stained with 4',6'‑diamidino‑2‑phenylindole. The results showed that GPATC2 is predominantly expressed in the nucleus of 293T cells. Overexpression of GPATC2 may inhibit transcription of the NF-κB reporter gene. The role of GPATC2 in proliferation was analyzed with cell counting kit-8, colony-forming efficiency and flow cytometry assays. The results indicated that over‑expression of GPATC2 in 293T cells significantly inhibited cell proliferation by decreasing the number of cells in S phase. By contrast, GPATC2 knockdown by RNA interference exhibited the opposite effect, suggesting that GPATC2 may be involved in inhibiting G1-S phase transition in 293T cells. In conclusion, these results provide novel insight into the breadth of expression of GPATC2 and its role in cell proliferation.

Maintenance and distribution of epithelial stem/progenitor cells after corneal reconstruction using oral mucosal epithelial cell sheets.

We assessed the maintenance and distribution of epithelial stem/progenitor cells after corneal reconstruction using tissue-engineered oral mucosal cell sheets in a rat model. Oral mucosal biopsy specimens were excised from green fluorescent protein (GFP) rats and enzymatically treated with Dispase II. These cells were cultured on inserts with mitomycin C-treated NIH/3T3 cells, and the resulting cell sheets were harvested. These tissue-engineered cell sheets from GFP rats were transplanted onto the eyes of a nude rat limbal stem cell deficiency model. Eight weeks after surgery, ocular surfaces were completely covered by the epithelium with GFP-positive cells. Transplanted corneas expressed p63 in the basal layers and K14 in all epithelial layers. Epithelial cells harvested from the central and peripheral areas of reconstructed corneas were isolated for a colony-forming assay, which showed that the colony-forming efficiency of the peripheral epithelial cells was significantly higher than that of the central epithelial cells 8 weeks after corneal reconstruction. Thus, in this rat model, the peripheral cornea could maintain more stem/progenitor cells than the central cornea after corneal reconstruction using oral mucosal epithelial cell sheets.

Re-characterization of established human retinoblastoma cell lines.

Retinoblastoma (RB) is the most common malignant intraocular childhood tumor. Forty years after their first description, in the present study, we re-characterized seven established retinoblastoma cell lines with regard to their RB1 mutation status, morphology, growth pattern, endogenous apoptosis levels, colony formation efficiency in soft agar and invasiveness and dissemination capacity in chick chorioallantoic membrane (CAM) assays. All RB cell lines predominantly resemble small epithelioid cells with little cytoplasm and large nucleus, which mainly grow in cell clusters, but sometimes form chain-like structures with incident loops or three-dimensional aggregates. We observed different growth rates for the different retinoblastoma cells investigated. RBL-30, RBL-13 and RBL 383 cells grew very slowly, whereas Y-79 cells grew fastest under our culture conditions. Apoptosis rates likewise differed with highest cell death levels in RB 383 and RB 355 and lowest in WERI-Rb1 and RBL-15. Contradicting former reports, six of the seven RB cell lines analyzed were able to form colonies in soft agarose after single cell seeding within 3 weeks of incubation. Upon inoculation of four out of seven RB cell lines on the dorsal CAM, GFP-positive cells were detectable in the ventral CAM and two RB cell lines caused tumor development, indicating their intravasation and dissemination potential. All RB cell lines exhibited the potential to extravasate from the capillary system after intravenous CAM injection. Our study provides valuable new details for future therapy-related retinoblastoma basic research in vitro.

Human Limbal Mesenchymal Cells Support the Growth of Human Corneal Epithelial Stem/Progenitor Cells

We tested the viability of human limbal mesenchymal cells (LMCs) to support the expansion of human corneal epithelial stem/progenitor cells (LSCs). Human LMCs were isolated from sclerocorneal tissue using collagenase A. Primary limbal epithelial cells (LECs) in the form of single cell suspension or cell clusters were cocultured on a monolayer of either 3T3 cells (control) or LMCs (SC-LMC culture). The LEC clusters also were grown directly on LMCs (CC-LMC culture) and in an optimized 3-dimensional culture method (3D CC-LMC culture). Colony-forming efficiency (CFE) and LEC proliferation were analyzed. The phenotype of the cultured LECs was assessed by their expression level of putative stem cell markers and a differentiation marker by qRT-PCR and immunocytochemistry. The LECs in the SC-LMC culture had a very limited growth and the stem/progenitor phenotype was lost compared to the control. Growth and cell morphology improved using the CC-LMC culture. The 3D CC-LMC culture method was the best to support the growth of the LSC population. Expression of ATP-binding cassette family G2 and ΔNp63 at the mRNA level was maintained or increased in CC-LMCs and 3D CC-LMC cultures compared to the control. The percentage of the K14(+) and K12(+) cells was comparable in these three cultures. There was no significant difference in the percentage of p63α high expressing cells in the control (21%) and 3D CC-LMC culture (17%, P > 0.05). Human LMCs can substitute 3T3 cells in the expansion of LSCs using the 3-dimensional culture system.

Abstract 62: Third hit genetic changes and clonal heterogeneity in a genetically engineered mouse lung cancer model

Abstract Background: Cancer cells may undergo mutations beyond their original causal changes, resulting in clonal expansion, and potentially driving tumor heterogeneity and evolution. Our hypothesis was that additional, “third hit” genetic changes in clonally derived tumors, originally generated by the same oncogenic stimulus, would have different phenotypes. Methods: Epithelial cells were isolated from LSL-KrasG12D+/-/Trp53fl/fl/Rosa-CAG-LSL-tdTomato+/- and C57BL/6 control mouse tracheas and were cultured on plastic. On day 3, cells were treated with an adenovirus expressing Cre recombinase. After 7 days, single EPCAM+, 7-aminoactinomycin D (7-AAD)-, and tdTomato+ cells from the LSL-KrasG12D+/-/Trp53fl/fl/Rosa-CAG-LSL-tdTomato+/- mouse, and EPCAM+/ 7-AAD- cells from the control mouse were FACS sorted, and individually seeded in 96-well plates. Clones developing in each well were passaged separately and characterized using growth curves on plastic, staining for lineage markers, and colony forming efficiency (CFE) and colony morphology in 3D culture. Selected clones were further investigated in vivo after subcutaneous injection into the flanks of NOD/SCID mice. Agilent SureSelect targeted exome sequencing was used to evaluate genetic variations in cells derived from each cell clone. Results: Thirty-five LSL-KrasG12D+/-/Trp53fl/fl/Rosa-CAG-LSL-tdTomato+/- transformed clones and 2 control mouse clones were serially passaged, and 4 transformed cell lines representing distinct phenotypes (named MT1, 2, 3, and 4), were identified based on growth rates, lineage marker staining, CFE, and 3D colony morphology. All 4 clones were pancytokeratin+, 3 were thyroid trasnscription factor-1 (TTF-1)+ and one was p63+. All 4 cell lines generated tumors in NOD/SCID mice, which exhibited variable histology and marker expression. Exome sequencing revealed unique copy number profiles. Specifically, MT1 had gains and losses in chromosome 3, MT2 had losses in chromosome 9, MT3 had losses in chromosomes 7 and 14, and MT4 had losses in chromosome 7. Mutations potentially representing third hits were identified in each cell line, particularly Ss18l1 (synovial sarcoma translocation gene on chromosome 18-like 1) and Gas7 (growth arrest specific 7) in MT1, Runx1 (runt related transcription factor 1) and Dicer1 (dicer 1, ribonuclease type III) in MT2, Apc (adenomatous polyposis coli) in MT3, and Csmd1 (CUB and Sushi multiple domains 1) in MT4. Conclusions: Multiple single cell derived mouse tracheal epithelial tumor cell clones were generated from a single LSL-KrasG12D+/-/Trp53fl/fl/Rosa-CAG-LSL-tdTomato+/- mouse. Selected clones exhibited unique growth rates, lineage marker expression patterns, CFE, 3D cell morphology and variable in vivo tumor histology and marker expression. Unique copy number aberrations and third hit mutations likely generate distinct clone phenotypes, driving tumor heterogeneity and evolution. Citation Format: Sang-Won Um, Ping-Jie Xiao, David Neil Hayes, William Y. Kim, Scott H. Randell. Third hit genetic changes and clonal heterogeneity in a genetically engineered mouse lung cancer model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 62. doi:10.1158/1538-7445.AM2014-62

Abstract 4866: Enhanced adipogenesis of bone marrow-derived stromal cells by ovarian cancer cells via BMP4 and IL8 is associated with increase in cancer stem/progenitor cells and cancer cell proliferation

Abstract Collaborative interactions between malignant cancer cells and their surrounding nonmalignant stromal cells are increasingly recognized as crucial factors in tumor biology. In adenocarcinomas such as serous ovarian cancer, stromal cells in the microenvironment are recruited either locally from neighboring tissues or distantly from the circulation and bone marrow. To study the effects of bone marrow-derived stromal cells on ovarian cancer, two bone marrow preadipocyte stromal cell lines, MS5 and OP9, were used in coculture with ovarian cancer cell lines. Large number of adipocytes was observed in coculture of ovarian cancer cells with stromal cells when compared to stromal cells cultured alone. To quantify the extent of adipogenesis induced by ovarian cancer cells, MS5 or OP9 was cocultured with three ovarian cancer cell lines OVCAR3, OVCAR5 and SKOV3 by using 0.4 μm transwell permeable support and stained for oil red O after 7 days. OVCAR5 coculture resulted in highest increase in the amount of oil red O stain of 2.5±0.6-fold in MS5 and 2.6±0.8-fold in OP9 compared to MS5 or OP9 alone. Recently, it was reported that adipocytes promote ovarian cancer growth by providing energy to cancer cells (Nieman et al. Nature Medicine, 2011). In support of this observation, we found that coculture of MS5 yielded a 1.9±0.1-fold increase in number of OVCAR5 cells. After coculturing with MS5 or OP9 stromal cells for 7 days, OVCAR5 cells were harvested from the transwell permeable support to allow colonies to form in low melting point agarose for 28 days to determine the number of cancer stem/progenitor cells. OVCAR5 cultured alone has a colony formation efficiency of 2.64%. OVCAR5 that was cocultured with MS5 or OP9 has a higher percentage of colonies of 6.52% and 9.2% respectively. A panel of 17 cytokines/growth factors was analyzed by RT-PCR to determine the putative factors secreted by OVCAR5 that promotes adipogenesis. BMP4 and IL8 were found to be highly expressed in OVCAR5 when compared to SKOV3, which only mildly increased adipocyte number. shRNAs against BMP4 and IL8 were used to silence the respective cytokines in OVCAR5 by lentiviral vectors. Coculture of shBMP4 or shIL8 OVCAR5 cells with MS5 in transwell filter each resulted in a 15 and 24% decrease in oil red O stain, confirming the enhanced adipogenesis was partly conferred by BMP4 and IL8 secreted by OVCAR5. Our results illustrate how ovarian tumor cells modulate the host environment by stimulating differentiation of adipocytes from their precursor cells from the bone marrow mesenchymal stem cells, which in turn favor the proliferation and increase the number of ovarian cancer stem cells. This study strengthens the importance of tumor microenvironment in providing a tumor stem cell niche and proposes possible targets for cancer therapy that counteracts the differentiation of adipocytes caused by ovarian cancer cells. Citation Format: Elissa Wai Pung Wong, Lingbo Shen, Jae-Hung Shieh, Malcolm A. S. Moore. Enhanced adipogenesis of bone marrow-derived stromal cells by ovarian cancer cells via BMP4 and IL8 is associated with increase in cancer stem/progenitor cells and cancer cell proliferation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4866. doi:10.1158/1538-7445.AM2014-4866

Roles of PIK3R1 gene in development of hepatocellular carcinoma

To investigate the roles of phosphatidylinositol 3 kinase regulatory subunit alpha (PIK3R1）gene in the development of hepatocellular carcinoma （HCC）. Surgical specimens of liver cancer and corresponding pericancerous liver tissue were collected from 20 patients with hepatocellular carcinoma. Expression of p85α, encoded by PIK3R1, in HCC tissue specimens was detected by Western blotting and immunohistochemistry. HCC HepG2 cells were transfected with PIK3R1 siRNA or PIK3R1-cDNA. The expression of PIK3R1 in transfected HepG2 cells or control cells were detected by real-time PCR. Cell proliferation was evaluated by MTT, colony formation assays and flow cytometry respectively. The expression of PI3K/AKT pathway-related proteins were detected by Western blotting. The expression of p85α in liver tissue was higher than that in pericancerous tissues (1.27±0.58 vs 0.99±0.47，t=-3.25，P<0.05）. The expression of PIK3R1 was decreased by 0.19±0.03 fold in PIK3R1siRNA-transfected HepG2 cells(t=46.77，P<0.05)，and increased by 32.36±3.33 fold in PIK3R1 cDNA -transfected cells（t=-16.31， P<0.05）. MTT result showed that PIK3R1 siRNA inhibited growth of HepG2 cells （0.611±0.072 vs 0.807±0.059，t=3.65，P<0.05），while PIK3R1 cDNA increased the cell growth（0.937±0.060 vs 0.693±0.065，t=-4.78，P<0.05）. PIK3R1 siRNA transfected cells presented lower colony-forming efficiency than control group（3.8%±0.84% vs 15.0%±2.3%，t=7.92，P<0.05），while PIK3R1 cDNA transfected cells had higher colony-forming efficiency than control group (23.6%±3.4% vs 12.0%±1.5%，t=-5.40，P<0.05）. PIK3R1 siRNA reduced the ratio of S phase cells（13.9%±0.015% vs 32.9%±0.07%，t=45.97，P<0.01, while PIK3R1 cDNA increased S phase cells（56.33%±0.024% vs 31.94%±0.042%，t=-8.73，P<0.01）. PIK3R1 increased the level of p-AKT and decreased p53 level. CONCLUSION：p85α is highly expressed in HCC，and PIK3R1 gene may promote proliferation of HepG2 cells by activating PI3K/AKT pathway.

Stem cells from apical papilla and their properties in two primary culture methods

Background and Objective: Stem cells from the apical papilla (SCAP) are a unique population of dental mesenchymal stem cells and also different to dental pulp stem cells. Study and application of this cell type are extremely important in natural tooth root regeneration, especially in the treatment of oral diseases. Studies have isolated and characterized stem cell properties of SCAP by only one culture method- enzymatic dispersal method. The purpose of this study was to compare two primary culture methods in isolation and characterization of SCAP.Materials and Methods: The effects of two methods on the cell viability, colony-forming efficiency, proliferation rate and multilineage differentiation potential and profiles of mesenchymal stem cell markers in vitro have been examined.Result: We found that cultured cells adhered to the surface and developed with the same proliferation rate and formed colony units in both methods. In the enzymatic dispersal method, time for primary culture is shorter, differentiation potential is also higher than in outgrowth method. However, the cultured population of cells by outgrowth method is more homogenous with high expression of CD44, CD73, CD90 and negative with CD34, CD45, HLADR (<2%).Conclusion: We conclude that two methods are available for primary culture of SCAP and in different situations, we should use more appropriate method.

Effect of interleukin-8 on migration and invasion of CD133 + positive liver cancer stem cells

Objective To study the effect and possible mechanism of interleukin (IL)-8 on migration and invasion of CD133 + liver cancer stem cells (LCSCs).Methods CD133 + cells were isolated by using magnetic-activated cell sorting (MACS) from MHCC97-H cells.Migration,invasion and colony formation efficiency of CD133 +/CD133-cells were detected.IL-8 in the supernatants was detected by enzyme linked immunosorbent assay (ELISA).Tumor-forming ability was observed by xenografts in the BALB/c nude mice.The effect of IL-8 neutalizing antibody on migration,invasion and colony formation efficiency of the sorted cells was determined.Results IL-8 was up-regulated in CD133 + cells [(85.76 ± 2.55) ng/L] as compared with CD133-cells [(13.40 ± 2.19) ng/L].The CD133 + cells exhibited a greater ability of colony formation efficiency [(13.50 ± 0.62) ％ vs.(4.43 ± 0.31) ％],migration [(39.3 ± 3.2) vs.(18.0 ± 2.6)],invasion [(37.5 ± 3.0) vs.(20.3 ± 1.8)] and tumors initiation.Colony formation efficiency was decreased in both CD133 + [(4.63 ± 0.47)％ vs.(12.93 ± 0.55)％] and CD133-[(3.53 ±0.49)％ vs.(6.67 ±0.15)％] cells treated by IL-8 neutralizing antibody.IL-8 neutralizing antibody suppressed the migration [(24.7 ± 2.8) vs.(37.7 ± 2.8)] and invasion [(25.2 ± 2.1) vs.(40.2 ± 3.6)] of the CD133 + cells specifically.Conclusion CD133 plays a functional part in regulating migration and invasion of LCSCs via regulating IL-8 signaling. Key words: Interleukin-8 ; CD133 ; Liver cancer stem cells ; Invasion; Migration

Effects of As2O3 in combination with TPA on K562 cells

This study was aimed to investigate the effects of arsenic trioxide (As2O3) combined with TPA on cell cycle, cell differentiation and apoptosis of K562 cell line, and their possible mechanisms. K562 cells were treated with 200 nmol/L TPA, 2 µmol/L As2O3 alone and 200 nmol/L TPA combined with 2 µmol/L As2O3. The proliferative inhibition rates were determined with CCK-8. Annexin V and agarose gel electrophoresis were adopted to detect apoptosis. Colony formation test was used to determine the colony-formation efficiency. Flow cytometry was used to detect the cell differentiation and cell cycle changes. Western blot was employed to detect the expression of P38 and p-P38 proteins. The results showed that combination treatment had synergistic effects on the proliferative inhibition and apoptosis, which were much higher than those treated alone. As2O3 could decrease the colony formation ability of K562 cells. The cells treated with both TPA and As2O3 expressed far more CD11b antigens compared with cells exposed to As2O3 alone. K562 cells treated with TPA were arrested in G1 phase compared with the control group, As2O3 increased the percentage of K562 cells in the G2 phase. The combination treatment increased the expression of p-P38 of K562 cells compared with the cells exposed As2O3 alone. It is concluded that TPA can enhance the effect of As2O3 on inducing apoptosis and adjusting cell cycle , which will expect to provide a new therapeutic program.

Single cell sorting identifies progenitor cell population from full thickness bovine articular cartilage

To date, no approved clinical intervention successfully prevents the progressive degradation of injured articular cartilage that leads to osteoarthritis (OA). Stem/progenitor cell populations within tissues of diarthrodial joint have shown their therapeutic potential in treating OA. However, this potential has not been fully realized due in part to the heterogeneity of these subpopulations. Characterization of clonal populations derived from a single cell may help identify more homogenous stem/progenitor populations within articular cartilage. Moreover, chondrogenic potential of clonal populations from different zones could be further examined to elucidate their differential roles in maintaining articular cartilage homeostasis. We combined Fluorescence-activated cell sorting (FACS) and clonogenicity screening to identify stem/progenitor cells cloned from single cells. High-efficiency colony-forming cells (HCCs) were isolated, and evaluated for stem/progenitor cell characteristics. HCCs were also isolated from different zones of articular cartilage. Their function was compared by lineage-specific gene expression, and differentiation potential. A difference in colony-forming efficiency was observed in terms of colony sizes. HCCs were highly clonogenic and multipotent, and overexpressed stem/progenitor cell markers. Also, proliferation and migration associated genes were over-expressed in HCCs. HCCs showed zonal differences with deep HCCs more chondrogenic and osteogenic than superficial HCCs. Our approach is a simple yet practical way to identify homogeneous stem/progenitor cell populations with clonal origin. The discovery of progenitor cells demonstrates the intrinsic self-repairing potential of articular cartilage. Differences in differentiation potential may represent the distinct roles of superficial and deep zone stem/progenitor cells in the maintenance of articular cartilage homeostasis.

Collecting duct-derived cells display mesenchymal stem cell properties and retain selective in vitro and in vivo epithelial capacity.

We previously described a mesenchymal stem cell (MSC)-like population within the adult mouse kidney that displays long-term colony-forming efficiency, clonogenicity, immunosuppression, and panmesodermal potential. Although phenotypically similar to bone marrow (BM)-MSCs, kidney MSC-like cells display a distinct expression profile. FACS sorting from Hoxb7/enhanced green fluorescent protein (GFP) mice identified the collecting duct as a source of kidney MSC-like cells, with these cells undergoing an epithelial-to-mesenchymal transition to form clonogenic, long-term, self-renewing MSC-like cells. Notably, after extensive passage, kidney MSC-like cells selectively integrated into the aquaporin 2-positive medullary collecting duct when microinjected into the kidneys of neonatal mice. No epithelial integration was observed after injection of BM-MSCs. Indeed, kidney MSC-like cells retained a capacity to form epithelial structures in vitro and in vivo, and conditioned media from these cells supported epithelial repair in vitro. To investigate the origin of kidney MSC-like cells, we further examined Hoxb7(+) fractions within the kidney across postnatal development, identifying a neonatal interstitial GFP(lo) (Hoxb7(lo)) population displaying an expression profile intermediate between epithelium and interstitium. Temporal analyses with Wnt4(GCE/+):R26(tdTomato/+) mice revealed evidence for the intercalation of a Wnt4-expressing interstitial population into the neonatal collecting duct, suggesting that such intercalation may represent a normal developmental mechanism giving rise to a distinct collecting duct subpopulation. These results extend previous observations of papillary stem cell activity and collecting duct plasticity and imply a role for such cells in collecting duct formation and, possibly, repair.