Evaluation of unrestricted somatic stem cells as a feeder layer to support undifferentiated embryonic stem cells

Abstract

The use of unrestricted somatic stem cells (USSCs) holds great promise for future clinical applications. Conventionally, mouse embryonic fibroblasts (MEFs) or other animal-based feeder layers are used to support embryonic stem cell (ESC) growth; the use of such feeder cells increases the risk of retroviral and other pathogenic infection in clinical trials. Implementation of a human-based feeder layer, such as hUSSCs that are isolated from human sources, lowers such risks. Isolated cord blood USSCs derived from various donors were used as a novel, supportive feeder layer for growth of C4mES cells (Royan C4 ESCs). Complete cellular characterization using immunocytochemical and flow cytometric methods were performed on murine ESCs (mESCs) and hUSSCs. mESCs cultured on hUSSCs showed similar cellular morphology and presented the same cell markers of undifferentiated mESC as would have been observed in mESCs grown on MEFs. Our data revealed these cells had negative expression of Stat3, Sox2, and Fgf4 genes while showing positive expression for Pou5f1, Nanog, Rex1, Brachyury, Lif, Lifr, Tert, B2m, and Bmp4 genes. Moreover, mESCs cultured on hUSSCs exhibited proven differentiation potential to germ cell layers showing normal karyotype. The major advantage of hUSSCs is their ability to be continuously cultured for at least 50 passages. We have also found that hUSSCs have the potential to provide ESC support from the early moments of isolation. Further study of hUSSC as a novel human feeder layer may lead to their incorporation into clinical methods, making them a vital part of the application of human ESCs in clinical cell therapy.

Unrestricted somatic stem cells differentiation into hepatic-like cells in a three dimensional arrangement

“…Moreover, human umbilical cord blood (CB)-derived USSCs have previously been proved to have a vast differentiation potential and regenerative benevolent effects when applied in animal models of multiple degenerative diseases. It has also been suggested that CBderived USSCs could significantly contribute in wound healing and be potentially used in cell therapy in some genetic skin diseases including recessive dystrophic epidermolysisbullosa (10,11).…”

“…Somatic stem cells are distinct but capable to differentiate into mesenchymal cells (bone cells, chondrocytes), endodermal cells, neural cells, but not into endothelial stem or progenitor cells and hematopoietic lineage stem or progenitor cells and adipocytes characterized in that the USSC expresses the DLKl -gene the Arvand J Health Med Sci 2016;1(1):17-26 expression being preferably detectable and measurable using RT-PCR assay and/or on the protein level and that it is a clonally selected and expanded cell (10,16,17). Unrestricted adherent pluripotent isolated cells from umbilical cord blood (USSCs) have the capability to differentiate into osteoblasts, blood cells, chondrocytes, neurons, hepatocytes, and heart tissue under ex vivo conditions (10,17). USSCs express different factors, including growth factors, adhesion molecules and various cytokines such as SCF, TGF1b, IL-6, G-CSF, VEGF, GM-CSF, M-CSF, LIF, Flt3 ligand, TPO, SDF-1a, IL-15, IL-12, HGF, IL-8, and IL1b (10).…”

“…Unrestricted adherent pluripotent isolated cells from umbilical cord blood (USSCs) have the capability to differentiate into osteoblasts, blood cells, chondrocytes, neurons, hepatocytes, and heart tissue under ex vivo conditions (10,17). USSCs express different factors, including growth factors, adhesion molecules and various cytokines such as SCF, TGF1b, IL-6, G-CSF, VEGF, GM-CSF, M-CSF, LIF, Flt3 ligand, TPO, SDF-1a, IL-15, IL-12, HGF, IL-8, and IL1b (10). They can also be applied as a reliable feeder layer.…”

“…CB-derived stem cells have higher telomere length and proliferation capacities compared to bone marrow derived MSC which is accompanied by a lower exposure to pathogens until extraction (9)(10)(11). CB-MSC and unrestricted somatic stem cells (USSC) share many overlapping features concerning the immunophenotype and differentiation potential.…”

“…We were recognized for the appropriate usage of human cord blood by the ethical and safety committee of the Shahid Beheshti University of Medical Sciences. Isolation and characterization of human USSCs was performed as described in previous studies (10,11,17). Briefly with some modifications, human cord blood was collected from 30 informed consent mother's umbilical cord vein.…”