Background: Small molecule compounds have been well recognized for their promising power in generation, expansion and maintenance of embryonic or adult stem cells. The aim of this study was to identify a novel combination of small molecules in order to optimize the ex vivo expansion of umbilical cord blood derived-CD34+ cells. Methods: Considering the most important signaling pathways involved in the self-renewal of hematopoietic stem cells, CB-CD34+ cells were expanded with cytokines in the presence of seven small molecules including SB, PD, Chir, Bpv, Pur, Pμ and NAM. Eliminativism approach was used to find the best combination of selected small molecules for effective ex vivo expansion of CD34+ cell. In each step, proliferation, self-renewal, and clonogenic potential of the expanded cells as well as expression of some hematopoietic stem cell related genes were studied. Finally, the engraftment potential of expanded cells was also examined by the mouse intra-uterine transplantation model. Results: Our data shows that simultaneous use of SB431542 (TGF-β inhibitor), Chir9901 (GSK3 inhibitor) and Bpv (PTEN inhibitor), resulted in a 50-fold increase in the number of CD34+CD38- cells. This was further reflected in approximately 3 times increase in clonogenic potential of the small molecule cocktail-expanded cells. These cells, also, showed a 1.5-fold higher engraftment potential in the peripheral blood of NMRI model of in utero transplantation. These results are in total conformity with up-regulation of HOXB4, GATA2 and CD34 marker gene as well as CXCR4 homing gene. Conclusion: Taken together, our findings introduce a novel combination of small molecules to improve the yield of existing protocols used in the expansion of hematopoietic stem cells.

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Objective One of the main approaches to preventing skin ageing is to protect fibroblast cells from oxidative stress. The promoting effect of the human amniotic membrane extract (hAME) on re‐epithelization, proliferation and migration of cells in wound healing has been already well studied. This experimental study aimed to investigate the antioxidant activity of hAME against hydrogen peroxide (H2O2)‐induced dermal fibroblast damage. Methods Here, to establish the ageing model, human foreskin fibroblasts (HFFs) were exposed to 200 μM H2O2 for 2 h. HFFs were treated with 0.1 mg/ml AME for 24 or 48 h before or/and after H2O2 exposure. A total of 48 h following the H2O2 treatment, we measured cell proliferation, viability, senescence‐associated β‐galactosidase (SA‐β‐Gal), antioxidants and preinflammatory cytokine (IL‐6) levels, as well as the expression of senescence‐associated genes (P53 and P21). Results The obtained results indicated that under oxidative stress, AME significantly increased cellular viability and not only promoted the cell proliferation rate but also attenuated apoptotic induction condition (p < 0.001). AME also significantly reversed the SA‐β‐Gal levels induced by H2O2 (p < 0.001). Additionally, both pre‐ and post‐treatment regimen by AME down‐regulated the expression of senescence marker genes (p < 0.001). Moreover, AME declined different oxidative stress biomarkers such as superoxide dismutase and catalase and increased the glutathione amount. Conclusion Altogether, our results indicated that AME had a remarkable antioxidant and antiageing activity as pre‐ and post‐treatment regimen, pointing to this compound as a potential natural‐based cosmeceutical agent to prevent and treat skin ageing conditions.

Abstract Background: Small molecule compounds have been well recognized for their promising power in generation, expansion and maintenance of embryonic or adult stem cells. The aim of this study was to identify a novel combination of small molecules in order to optimize the ex vivo expansion of umbilical cord blood derived-CD34+ cells. Methods: Considering the most important signaling pathways involved in the self-renewal of hematopoietic stem cells, CB-CD34+ cells were expanded with cytokines in the presence of seven small molecules including SB, PD, Chir, Bpv, Pur, Pμ and NAM. Eliminativism approach was used to find the best combination of selected small molecules for effective ex vivo expansion of CD34+ cell. In each step, proliferation, self-renewal, and clonogenic potential of the expanded cells as well as expression of some hematopoietic stem cell related genes were studied. Finally, the engraftment potential of expanded cells was also examined by the mouse intra-uterine transplantation model. Results: Our data shows that simultaneous use of SB431542 (TGF-β inhibitor), Chir9901 (GSK3 inhibitor) and Bpv (PTEN inhibitor), resulted in a 50-fold increase in the number of CD34+CD38- cells. This was further reflected in approximately 3 times increase in clonogenic potential of the small molecule cocktail-expanded cells. These cells, also, showed a 1.5-fold higher engraftment potential in the peripheral blood of NMRI model of in utero transplantation. These results are in total conformity with up-regulation of HOXB4, GATA2 and CD34 marker gene as well as CXCR4 homing gene. Conclusion: Taken together, our findings introduce a novel combination of small molecules to improve the yield of existing protocols used in the expansion of hematopoietic stem cells.