Selection of alkaline phosphatase-positive induced pluripotent stem cells from human amniotic fluid-derived cells by feeder-free system

Introduction. Pluripotent stem cells are believed to have greater clinical potential than mesenchymal stem cells due to their ability to differentiate into almost any cell type of an organism, and since 2006, the generation of patient-specific induced pluripotent stem cells (iPSCs) has become possible in multiple species. Objectives. We hypothesize that different cell types respond differently to the reprogramming process; thus, the goals of this study were to isolate and characterize equine adult and fetal cells and induce these cells to pluripotency for future regenerative and translational purposes. Methods. Adult equine fibroblasts (eFibros) and mesenchymal cells derived from the bone marrow (eBMmsc), adipose tissue (eADmsc), and umbilical cord tissue (eUCmsc) were isolated, their multipotency was characterized, and the cells were induced in vitro into pluripotency (eiPSCs). eiPSCs were generated through a lentiviral system using the factors OCT4, SOX2, c-MYC, and KLF4. The morphology and in vitro pluripotency maintenance potential (alkaline phosphatase detection, embryoid body formation, in vitro spontaneous differentiation, and expression of pluripotency markers) of the eiPSCs were characterized. Additionally, a miRNA profile analysis of the mesenchymal and eiPSCs was performed. Results. Multipotent cells were successfully isolated, but the eBMmsc failed to generate eiPSCs. The eADmsc-, eUCmsc-, and eFibros-derived iPSCs were positive for alkaline phosphatase, OCT4 and NANOG, were exclusively dependent on bFGF, and formed embryoid bodies. The miRNA profile revealed a segregated pattern between the eiPSCs and multipotent controls: the levels of miR-302/367 and the miR-92 family were increased in the eiPSCs, while the levels of miR-23, miR-27, and miR-30, as well as the let-7 family were increased in the nonpluripotent cells. Conclusions. We were able to generate bFGF-dependent iPSCs from eADmsc, eUCmsc, and eFibros with human OSKM, and the miRNA profile revealed that clonal lines may respond differently to the reprogramming process.

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35. Long-Term Engraftment and In Vivo Selection of Hematopoietic Stem/Progenitor Cells Generated By Vascular Niche Induction of Induced Pluripotent Stem Cells

Molecular Therapy ◽

10.1016/s1525-0016(16)35048-1 ◽

2014 ◽

Vol 22 ◽

pp. S14

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Progenitor Cells ◽

Pluripotent Stem Cells ◽

Hematopoietic Stem ◽

In Vivo Selection ◽

Induced Pluripotent ◽

Selection Of

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Trisomy 21 Mid-Trimester Amniotic Fluid Induced Pluripotent Stem Cells Maintain Genetic Signatures During Reprogramming: Implications for Disease Modeling and Cryobanking

Cellular Reprogramming ◽

10.1089/cell.2013.0091 ◽

2014 ◽

Vol 16 (5) ◽

pp. 331-344 ◽

Cited By ~ 7

Author(s):

Caterina Pipino ◽

Sayandip Mukherjee ◽

Anna L David ◽

Michael P. Blundell ◽

Steven W. Shaw ◽

...

Keyword(s):

Stem Cells ◽

Amniotic Fluid ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Trisomy 21 ◽

Disease Modeling ◽

Induced Pluripotent ◽

Genetic Signatures

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Exogenous LIN28 Is Required for the Maintenance of Self-Renewal and Pluripotency in Presumptive Porcine-Induced Pluripotent Stem Cells

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.709286 ◽

2021 ◽

Vol 9 ◽

Author(s):

Warunya Chakritbudsabong ◽

Somjit Chaiwattanarungruengpaisan ◽

Ladawan Sariya ◽

Sirikron Pamonsupornvichit ◽

Joao N. Ferreira ◽

...

Keyword(s):

Stem Cells ◽

Alkaline Phosphatase ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Embryoid Bodies ◽

Self Renewal ◽

Fetal Fibroblasts ◽

Induced Pluripotent

Porcine species have been used in preclinical transplantation models for assessing the efficiency and safety of transplants before their application in human trials. Porcine-induced pluripotent stem cells (piPSCs) are traditionally established using four transcription factors (4TF): OCT4, SOX2, KLF4, and C-MYC. However, the inefficiencies in the reprogramming of piPSCs and the maintenance of their self-renewal and pluripotency remain challenges to be resolved. LIN28 was demonstrated to play a vital role in the induction of pluripotency in humans. To investigate whether this factor is similarly required by piPSCs, the effects of adding LIN28 to the 4TF induction method (5F approach) on the efficiency of piPSC reprogramming and maintenance of self-renewal and pluripotency were examined. Using a retroviral vector, porcine fetal fibroblasts were transfected with human OCT4, SOX2, KLF4, and C-MYC with or without LIN28. The colony morphology and chromosomal stability of these piPSC lines were examined and their pluripotency properties were characterized by investigating both their expression of pluripotency-associated genes and proteins and in vitro and in vivo differentiation capabilities. Alkaline phosphatase assay revealed the reprogramming efficiencies to be 0.33 and 0.17% for the 4TF and 5TF approaches, respectively, but the maintenance of self-renewal and pluripotency until passage 40 was 6.67 and 100%, respectively. Most of the 4TF-piPSC colonies were flat in shape, showed weak positivity for alkaline phosphatase, and expressed a significantly high level of SSEA-4 protein, except for one cell line (VSMUi001-A) whose properties were similar to those of the 5TF-piPSCs; that is, tightly packed and dome-like in shape, markedly positive for alkaline phosphatase, and expressing endogenous pluripotency genes (pOCT4, pSOX2, pNANOG, and pLIN28), significantly high levels of pluripotent proteins (OCT4, SOX2, NANOG, LIN28, and SSEA-1), and a significantly low level of SSEA-4 protein. VSMUi001-A and all 5F-piPSC lines formed embryoid bodies, underwent spontaneous cardiogenic differentiation with cardiac beating, expressed cardiomyocyte markers, and developed teratomas. In conclusion, in addition to the 4TF, LIN28 is required for the effective induction of piPSCs and the maintenance of their long-term self-renewal and pluripotency toward the development of all germ layers. These piPSCs have the potential applicability for veterinary science.

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