Human Mesenchymal Stem Cell Transplantation Improved Functional Outcomes Following Spinal Cord Injury Concomitantly with Neuroblast Regeneration

Purpose: Spinal cord injury (SCI) is damage to the spinal cord that resulted in irreversible neuronal loss, glial scar formation and axonal injury. Herein, we used the human amniotic fluid mesenchymal stem cells (hAF-MSCs) and their conditioned medium (CM), to investigate their ability in neuroblast and astrocyte production as well as functional recovery following SCI. Methods: Fifty-four adult rats were randomly divided into nine groups (n=6), included: Control, SCI, (SCI+DMEM), (SCI+CM), (SCI+MSCs), (SCI+Astrocyte), (SCI+Astrocyte+DMEM), (SCI+Astrocyte+CM) and (SCI+Astrocyte+MSCs). Following laminectomy and SCI induction, DMEM, CM, MSCs, and astrocytes were injected. Western blot was performed to explore the levels of the Sox2 protein in the MSCs-CM. The immunofluorescence staining against doublecortin (DCX) and glial fibrillary acidic protein (GFAP) was done. Finally, Basso-Beattie-Brenham (BBB) locomotor test was conducted to assess the neurological outcomes. Results: Our results showed that the MSCs increased the number of endogenous DCX-positive cells and decreased the number of GFAP-positive cells by mediating juxtacrine and paracrine mechanisms (P<0.001). Transplanted human astrocytes were converted to neuroblasts rather than astrocytes under influence of MSCs and CM in the SCI. Moreover, functional recovery indexes were promoted in those groups that received MSCs and CM. Conclusion: Taken together, our data indicate the MSCs via juxtacrine and paracrine pathways could direct the spinal cord endogenous neural stem cells (NSCs) to the neuroblasts lineage which indicates the capability of the MSCs in the increasing of the number of DCX-positive cells and astrocytes decline

The Inhibitory Effect of Ginger Extract on Ovarian Cancer Cell Line; Application of Systems Biology

This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from the authors or the publishers.Purpose: Ginger is a natural compound with anti-cancer properties. The effects of ginger and its mechanism on ovarian cancer and its cell line model, SKOV-3, are unclear. In this study, we have evaluated the effect of ginger extract on SKOV-3. Methods: SKOV-3 cells were incubated with ginger extract for 24, 48 and 72 hours. Cell toxicity assay was performed. Different data mining algorithms were applied to highlight the most important features contributing to ginger inhibition on the SKOV-3 cell proliferation. Moreover, Real-Time PCR was performed to assay p53, p21 and bcl-2 genes expression. For co-expression meta-analysis of p53, mutual ranking (MR) index and transformation to Z-values (Z distribution) were applied on available transcriptome data in NCBI GEO data repository. Results: The ginger extract significantly inhibited cancer growth in ovarian cancer cell line. The most important attribute was 60 μg/ml concentration which received weights higher than 0.50, 0.75 and 0.95 by 90%, 80% and 50% of feature selection models, respectively. The expression level of p53 was increased sharply in response to ginger treatment. Systems biology analysis and meta-analysis of deposited expression value in NCBI based on rank of correlation and Z-transformation approach unraveled the key co-expressed genes and coexpressed network of P53, as the key transcription factor induced by ginger extract. High co-expression between P53 and the other apoptosis-inducing proteins such as CASP2 and DEDD was noticeable, suggesting the molecular mechanism underpinning of ginger action. Conclusion: We found that the ginger extract has anticancer properties through p53 pathway to induce apoptosis

Generation of embryonic stem cell lines from IVF embryos and parthenotes

Efficient isolation and maintenance of pluripotent cell lines in livestock species have proven to be a major hurdle despite tremendous efforts over 3 decades. Little is known about the signalling pathways controlling pluripotency in mammals other than rodents and primates. To understand these mechanisms in the bovine due to a biomedical model for human diseases, biotechnology applications and commercial use, embryonic stem cells (ESCs) were isolated from IVF embryos in a conventional medium and in a medium with 3 small-molecule inhibitors (3i) which permit efficient self-renewal of ESCs in mice and rats. SU5402 inhibits the FGF receptor tyrosine kinases and PD184352 inhibits the ERK cascade. CHIR99021 inhibits GSK3 to increase ES-cell proliferation. The effect of three inhibitors “3i” on 1) the efficiency of ESC isolation and 2) maintenance of established bovine ESC lines (bESCs) was examined in the different media. Colony formation and attachment to the feeder layer differed among media and media containing the 3i components resulted in better growth and maintenance of putative ESCs compared with the others. Expression of 6 pluripotency related genes (Oct4, rex1, sox2, ssea1, alp and nanog) and four proteins (Oct4, SSEA1, SSEA4, Alkaline Phosphatase) were assessed. The results indicate that 3i can improve bovine ESC isolation and increase the pluripotent component of putative bovine ES cells. The second series of experiments investigated the generation of autologous cell lines. ES cells from parthenogenetically activated oocytes can provide autologous transplantable cells, which are immuno-compatible for the oocyte donors as well as an invaluable tool for genetic engineering for transplantation and epigenetic studies. We report the efficient isolation of 8 putative bovine parthenogenetic ESC lines in 3i medium from 15 in vitro produced parthenotes. The cells displayed pluripotency similar to IVF ESC lines and differentiated in suspension culture to form embryoid bodies (EBs) expressing markers of the three embryonic germ layers. The third series of experiments investigated the long-term maintenance of pluripotent bESCs during in vitro culture further and developed a robust method of cryopreservation which is essential requirements to maximize the use of ESCs in research and for biotechnology applications. 1) Eight putative bESC lines established in N2B27-3i medium and 1 established in conventional medium were cultured over 30 passages (> 240 days) and characterized at P23-30. All cell lines expressed markers of pluripotency. The cells formed EBs containing cells of the three embryonic germ layers. For cryopreservation, vitrification was compared with conventional slow-freezing. Survival rate and colony morphology of vitrified cell lines after thawing was significantly higher than after slow-freezing, (97% v/s 56% respectively; p<0.01). Cell lines survived and proliferated, with good morphology, following vitrification in both N2B27-3i, 95% (104/110) and conventional medium, 97% (93/96). Post-thawed vitrified cell lines were cultured for an additional 12 passages (P¬18+12, P19+12 and P23+12) and shown to retain pluripotency. The cells formed EBs in suspension culture and expressed genes representative of the three embryonic germ layers. Vitrification did not change the karyotype of cells. Finally, three cell lines isolated in conventional medium were maintained for 80-95 passages and vitrified. These cell lines also retained expression of pluripotent markers. In summary, the results in this thesis present 1) efficient derivation and characterization of pluripotent putative bESCs from IVF and parthenotes in 3i medium and 2) maintenance of cell lines in both conventional and 3i media which express pluripotent markers and retain the ability to differentiation in vitro after long-term culture, and 3) successful cryopreservation of cell lines, especially by vitrification. This thesis adds significantly to the knowledge on isolation, characterization and cryopreservation of pluripotent cell lines in livestock, especially the bovine

The Toxic Effect of Silibinin and Paclitaxel Combination on Endometrial Cancer Cell Line

Background & objectives: Among gynecologic malignancies, endometrial cancer is the fourth most frequent cause of cancer death all over the world. Paclitaxel is one of the chemotherapy regimens that is used against this cancer. Treatment of tumor with Paclitaxel induces apoptosis, but it is also associated with serious side effects. Thus, it is imperative to search for more effective and safer chemotherapeutic regimens. Silibinin is a milk thistle plant extract that its antioxidant effects against some cancers have been studied. The aim of this study was to examine the effect of Paclitaxel and Silibinin combination on endometrial cancer cell line (Hela). Methods: Hela cell line was cultured in 25cm2 flask in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). Then the numbers of live cells were calculated with trypan blue staining method and then the cells were seeded in to 96-well flat-bottomed culture plates and treated with Silibilin, Paclitaxel and Paclitaxel plus Silibilin together with the control without treatment. MTT assay was used to evaluate cytotoxicity of different treatments. Results: After 48 hours of treatment, Paclitaxel and Silibilin combination inhibited cell growth significantly compared with the other groups (p<0.05). Conclusions: It is indicated that combination of Paclitaxel and Silibilin can affect the growth arrest of Hela cancer cell line more&nbsp; effective than other treatments and is needed to be examined in vitro

Anti-Cancer Effect of Silibinin on Epithelial Ovarian Cancer Cell Line and P21 Gene Expression

Background & objectives: Epithelial ovarian carcinoma seems to be one of the most lethal cancer types among all gynecological malignancies. The conventional course of therapy includes chemotherapy. Actually most cancers respond to chemotherapy but in the long run drug resistance and side effects cause treatment failure. In addition, milk thistle (silibinin), a plant that has been used from ancient time because of its good effects on different organs, determined to have powerful antioxidant activity. &nbsp;The aim of this study was to examine the effect of silibinin on SKOV-3 cancer cell line after 48 hours of treatment and P21 gene expression which involves in cell cycle progression. Methods: The human epithelial ovarian cancer cell line SKOV-3 was cultured as monolayer in 25 cm2 flask in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). Then the numbers of live cells were calculated using hemocytometer method and the cells were seeded in 96-well flat-bottomed culture plates and treated with different concentration of Silibinin. MTT assay was carried out to determine cell viability. To study P21 gene expression, RNA extraction and cDNA synthesis were carried out and real-time PCR was done. Results: Cell growth was inhibited considerably by Silibinin treated groups compared with control after 48 hours. P21 gene expression was increased as well. Conclusions: According to the results, Silibinin can be used as an effective drug in cancer treatment. More studies on animal models are also suggested

The Restorative Effect of Human Amniotic Fluid Stem Cells on Spinal Cord Injury

Spinal cord injury (SCI) is a debilitating condition within the neural system which is clinically manifested by sensory-motor dysfunction, leading, in some cases, to neural paralysis for the rest of the patient’s life. In the current study, mesenchymal stem cells (MSCs) were isolated from the human amniotic fluid, in order to study their juxtacrine and paracrine activities. Flow cytometry analysis was performed to identify the MSCs. A conditioned medium (CM) was collected to measure the level of BDNF, IL-1β, and IL-6 proteins using the ELISA assay. Following the SCI induction, MSCs and CM were injected into the lesion site, and also CM was infused intraperitoneally in the different groups. Two weeks after SCI induction, the spinal cord samples were examined to evaluate the expression of the doublecortin (DCX) and glial fibrillary acid protein (GFAP) markers using immunofluorescence staining. The MSCs’ phenotype was confirmed upon the expression and un-expression of the related CD markers. Our results show that MSCs increased the expression level of the DCX and decreased the level of the GFAP relative to the injury group (p &lt; 0.001). Additionally, the CM promoted the DCX expression rate (p &lt; 0.001) and decreased the GFAP expression rate (p &lt; 0.01) as compared with the injury group. Noteworthily, the restorative potential of the MSCs was higher than that of the CM (p &lt; 0.01). Large-scale meta-analysis of transcriptomic data highlighted PAK5, ST8SIA3, and NRXN1 as positively coexpressed genes with DCX. These genes are involved in neuroactive ligand–receptor interaction. Overall, our data revealed that both therapeutic interventions could promote the regeneration and restoration of the damaged neural tissue by increasing the rate of neuroblasts and decreasing the astrocytes

Isolation, Characterization, Cryopreservation of Human Amniotic Stem Cells and Differentiation to Osteogenic and Adipogenic Cells - Fig 4

<p>A and D) total population of cells are presented and highlighted population are transferred and tested for different markers, B) CD90-EP positive cells, C) CD44-FITC positive cells, and E) CD45 negative cells and F) C31 negative cells population.</p

Relative expression of Oct4 and NANOG at early and late passages.

<p>Expression level of Oct4 and NANOG were high at P5 (early passage) compared with P7 (late passage) (P< 0.05).</p

The doubling time of HAFSCs in different passages.

<p>As shown in this figure, by increasing the number of passages, the time of doubling time were increased. So, the doubling time related to P10 is significantly more than P7 and P4. All p-value were significant and p-value for P10 vs P7, P10 vs P4 and P7 vs P4 were .000.</p