فهرست مطالب zahra ghezelayagh

Efficient production of functional and mature alveolar epithelial is a major challenge for developing any cellreplacement therapy for lung degenerative diseases. The extracellular matrix (ECM) pro-vides a dynamic environmentand mediates cellular responses during development and maintenance of tissue functions. The decellularized ECM(dECM) which retains its native-like structure and bio-chemical composition can provide the induction of embryonicstem cell (ESC) differentiation toward the tissue-specific lineages during in vitro culture. Therefore, the aim of this studywas to evaluate the effect of sheep lung dECM-derived scaffold on differentiation and further maturation of ESC-derivedlung progenitor cells.

This study was an experimental study. In the first step, a sheep lung was decellularizedto achieve dECM scaffolds and hydrogels. Afterwards, the obtained dECM scaffold was evaluated for collagen andglycosaminoglycan contents, DNA quantification, and its ultrastructure. Next, the three experimental groups: i. Sheeplung dECM-derived scaffold, ii. Sheep lung dECM-derived hydrogel, and iii. Fibronectin-coated plates were comparedin their abilities to induce further differentiation of human embryonic stem cells (hESCs)-derived definitive endoderm(DE) into lung progenitor cells. The comparison was evaluated by immuno-staining and real-time polymerase chainreaction (PCR) assessments.

We found that the dECM-derived scaffold preserved its composition and native porous structures whilelacking nuclei and intact cells. All experimental groups displayed lung progenitor cell differen-tiation as revealed by theRNA and protein expression of NKX2.1, P63 and CK5. DE cells differenti-ated on dECM-derived scaffold and dECMderivedhydrogel showed significant upregulation of SOX9 gene expression, a marker of the distal airway epithelium.DE cells differentiated on the dECM-derived scaffold compared to the two other groups, showed enhanced expressionof SFTPC (type 2 alveolar epithelial [AT2] cell marker), FOXJ1 (ciliated cell marker), and MUC5A (secretory cell marker)genes.

Overall, our results suggest that dECM-derived scaffold improves the differentiation of DE cells towardslung alveolar progenitor cells in comparison with dECM-derived hydrogel and fibronectin-coated plates.

Growth factors are key elements of embryonic stem cell (ESC) research. Cell line development in eukaryotes is a time-consuming procedure which usually takes 12-18 months. Here, we report an easy and fast method with which production of Chinese hamster ovary (CHO) cells that express and secrete recombinant Activin A, as a major growth factor in endo/mesoderm differentiation of embryonic stem cells is achieved within 3-4 weeks.

In this experimental study, we cloned human Activin A into the pDONR/Zeo gateway entry vector using the BP reaction. Activin A was subcloned next into the pLIX_403 and pLenti6.3/TO/V5-DEST destination vectors by the LR reaction. The result was the production of constructs with which 293T cells were finally transfected for virus production. CHO cells were transduced using viral particles to produce a cell line that secretes the His6- Activin A fusion protein.

We developed a quick protocol which saves up to 3-4 weeks of time for producing recombinant proteins in CHO cells. The recombinant cell line produced 90 mg/L of functional Activin A measured in human ESC line Royan H5 (RH5), during in vitro differentiation into meso-endoderm and definitive endoderm.

Our results showed no significant differences in functionality between commercial Activin A and the one produced using our novel protocol. This approach can be easily used for producing recombinant proteins in CHO.