title: Differentiation of Definitive Endoderm from Human Induced Pluripotent Stem Cells on hMSCs Feeder in a Defined Medium.
abstract: BACKGROUND
  The Definitive Endoderm (DE) differentiation using the undefined media and non-human feeders can cause contaminations in the generated cells for therapeutic applications. Therefore, generating safer and more appropriate DE cells is needed. This study compared five different methods to establish an appropriate method for inducing an efficient DE differentiation from Human Induced Pluripotent Stem Cells (hiPSCs) on an appropriate feeder in a more defined medium.
  
  
  METHODS
  Human Induced Pluripotent Stem Cells (hiPSCs) were cultured on inactivated feeders. Passaged hiPSCs, without feeder, were incubated for three days with Activin-A and different endodermal differentiation media including 1-FBS, 2-B27, 3-ITS and albumin fraction-V, 4-B27 and ITS and 5-like the third medium. The feeder cells in the first four methods were Mouse Embryonic Fibroblasts (MEFs) and in the fifth method were human adult bone marrow Mesenchymal Stem Cells (hMSCs). DE markers FOXA2, SOX17 and CXCR4 and also pluripotency marker OCT4 were evaluated using qRT-PCR, as well as FOXA2 by the immunocytochemistry.
  
  
  RESULTS
  QRT-PCR analysis showed that after three days, the expression levels of DE and pluripotency markers in the differentiated hiPSCs among all five groups did not have any significant differences. Similarly, the immunocytochemistry analysis demonstrated that the differentiated hiPSCs expressed FOXA2, with no significant differences.
  
  
  CONCLUSIONS
  Despite this similarity in the results, the third differentiation medium has more defined and cost effective components. Furthermore, hMSC, a human feeder, is safer than MEF. Therefore, the fifth method is preferable among other DE differentiation methods and can serve as a fundamental method helping the development of regenerative medicine.
authors: Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
authors: Zahra
authors: Z
authors: Jaafarpour
authors: Department of Hematology, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran.
authors: Masoud
authors: M
authors: Soleimani
authors: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors: Saman
authors: S
authors: Hosseinkhani
authors: Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
authors: Mohammad Hossein
authors: MH
authors: Karimi
authors: Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
authors: Parichehreh
authors: P
authors: Yaghmaei
authors: Metabolic Disorders Research Center, Faculty of Medicine, Golestan University of Medical Sciences, Gorgan, Iran.
authors: Naser
authors: N
authors: Mobarra
authors: Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
authors: Bita
authors: B
authors: Geramizadeh
keywords: Endoderm; Induced pluripotent stem cells; Mesenchymal stem cells