Stem Cells from Human Exfoliated Deciduous Tooth Exhibit Stromal-Derived Inducing Activity and Lead to Generation of Neural Crest Cells from Human Embryonic Stem Cells

Objective: The neural crest is a transient structure of early vertebrate embryos that generates neural crest cells (NCCs). These cells can migrate throughout the body and produce a diverse array of mature tissue types. Due to the ethical and technical problems surrounding the isolation of these early human embryo cells, researchers have focused on in vitro studies to produce NCCs and increase their knowledge of neural crest development. Materials and Methods: In this experimental study, we cultured human embryonic stem cells (hESCs) on stromal stem cells from human exfoliated deciduous teeth (SHED) for a two-week period. We used different approaches to characterize these differentiated cells as neural precursor cells (NPCs) and NCCs. Results: In the first co-culture week, hESCs appeared as crater-like structures with marginal rosettes. NPCs derived from these structures expressed the early neural crest marker p75 in addition to numerous other genes associated with neural crest induction such as SNAIL, SLUG, PTX3 and SOX9. Flow cytometry analysis showed 70% of the cells were AP2/P75 positive. Moreover, the cells were able to self-renew, sustain multipotent differentiation potential, and readily form neurospheres in suspension culture. Conclusion: SHED, as an adult stem cell with a neural crest origin, has stromal-derived inducing activity (SDIA) and can be used as an NCC inducer from hESCs. These cells provide an invaluable resource to study neural crest differentiation in both normal and disordered human neural crest development

Generation of motor neurons by coculture of retinoic acid-pretreated embryonic stem cells with chicken notochords.

Understanding neuroectoderm formation and its subsequent diversification to functional neural subtypes remains elusive. We have shown here for the first time that embryonic stem cells (ESCs) can differentiate into neurons and motor neurons (MNs) by using a coculture embryonic notochord model in vitro. Mouse ESCs were induced to form neural precursors via timed exposure to retinoic acid (RA) using the 4-/4+ RA protocol. These cells were then cocultured with alginate bead-encapsulated notochords isolated from Hamburger and Hamilton stage 6-10 chick embryos. The use of notochord alone was not able to induce neural differentiation from ESCs, and, therefore, notochord does not possess neural inducing activity. Hence, the most successful neuronal cells and MN differentiation was only observed following the coculture of RA-pretreated ESCs with notochord. This resulted in a significantly greater number of cells expressing microtubule-associated protein-2 (MAP2), HB9, choline acetyltransferase (ChAT) and MN-specific genes. While further characterization of these differentiated cells will be essential before transplantation studies commence, these data illustrate the effectiveness of embryonic notochord coculture in providing valuable molecular cues for directed differentiation of ESCs toward an MN lineage

CLONING OF MOUSE PPARγ1 CDNA IN PEGFP-C1 EXPRESSION VECTOR

Peroxisome proliferated activated receptor g type 1 contains various functions in the cells including, regulation of cell growth and development, regulation of immune responses and energy homeostasis and regulation of stem cell differentiations. The aim of this study was to clone PPARg1 cDNA in a mammalian expression vector in a chimeric cDNA type, encompassing PPARg1 cDNA with EGFP cDNA for further transfection into the stem cells to study the role of PPARg1 in the process of stem cell differentiations. At the first step, total RNA was extracted from fat tissue of an adult mouse. Using specific primers PPARg1 cDNA was amplified to produce the entire length of ORF. RT-PCR products containing PPARg1 cDNA were treated by enzymatic digestion and inserted into the pEGFP-C1 downstream the EGFP cDNA and were used for transformation into bacterial competent cells. The positive colonies which showed inserted PPARg1 cDNA were selected for plasmid preparations and additional analysis performed to ensure that PPARg1 cDNA was inserted properly. Our results from enzymatic digestion and sequencing confirmed as it was expected, PPARg1 cDNA was amplified and cloned correctly. This cDNA gene encompasses 1428 bp

Creation of Tenecteplase-Producing CHO Cell Line Using Site-Specific Integrase from the Phage φC31

Objective: The aim of this study was to produce a stable CHO cell line expressing tenecteplase.Materials and Methods: In the first step, the tenecteplase coding sequence was clonedin a pDB2 vector containing attB recognition sites for the phage φC31 integrase. Then,using lipofection, the CHO cells were co-transfected with constructed recombinant plasmidencoding tenecteplase and attB recognition sites and the integrase coding sequencecontaining pCMV-Int plasmid. As the recombinant plasmid contained the neomycin resistancegene (neo), stable cells were then selected using G418 as an antibiotic. Stabletransformed cells were assessed using genomic PCR and RT-PCR. Finally, the functionalityof tenecteplase was evaluated on the cell culture media.Results: our results indicated that tenecteplase coding sequence was inserted into theCHO cell genome and was successfully expressed. Moreover, tenecteplase activity assessmentindicated the presence of our functional tenecteplase in the cell culture medium.Conclusion: Considering the data obtained from this study, φC31 integrase can be usedfor the production of a stable cell line and it be used to introduce ectopic genes into mammaliancells