Bioluminescence reporter gene imaging characterize human embryonic stem cell‐derived teratoma formation

Abstract

Human embryonic stem (hES) cells are capable of differentiation into virtually all cell types and hold tremendous potential as cell sources for regenerative therapies. However, teratoma formation can be the main obstacle for hES cells therapy. In order to understand the biology and physiology of hES cells teratoma formation, we investigated the angiogenic process within teratomas and characterized teratoma cells. In this study, hES cells transduced with double fusion reporter gene that consists of firefly luciferase and enhanced green fluorescent protein (Fluc-eGFP) were injected into hind limbs of SCID mice and performed longitudinal bioluminescence imaging on these animals. To test angiogenic contribution of teratoma from host or hES cells, human and mouse endothelial cells marker CD31 was stained respectively. To further explore the characterization of teratoma derived cells, flow cytometry analysis was carried out and GFP+/SSEA-4+ cells were isolated and subcultured. Then, we re-injected the isolated GFP+/SSEA-4+ teratoma cells into SCID mice and observed by imaging. Our results show that the reporter gene imaging is an ideal technology for monitoring long-term stem cell viability, death, and proliferation. Teratomas contained vasculatures are from hES cells and host. hESCs derived teratomas express a high level of undifferentiated marker SSEA-4 and CD56, and subcultured GFP+/SSEA-4+ cells had similar expression pattern comparing to undifferentiated hES cells, except for a very high level of CD56 and a little lower expression of undifferentiated markers, such as SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Moreover, the SSEA-4+ teratoma cells can form teratomas in SCID mice, and this type teratomas grow at a lower rate compared to teratomas derived from hES cells, and are more differentiated.

In vivo directed differentiation of pluripotent stem cells for skeletal regeneration

“…S2) (16). h-iPSCs were transduced with a firefly luciferase/ red fluorescent protein/herpes simplex virus thymidine kinase triple-fusion reporter construct and h-ESCs were labeled with a firefly luciferase/green fluorescent protein construct to follow in vivo cell viability (17). With both cell types, there was a significant increase in luciferase imaging over time consistent with biologic activity and teratoma formation (Fig.…”

124I-PET Assessment of Human Sodium Iodide Symporter Reporter Gene Activity for Highly Sensitive In Vivo Monitoring of Teratoma Formation in Mice

“…However, as 100 % purity cannot be achieved as of yet, the side effect of teratoma formation has to be considered. Indeed, teratoma formation as a side effect of non-differentiated cells in the applied cell preparation has been reported in a number of preclinical studies [17]. Therefore, in addition to further improvement of purification technologies, the clinical use of stem cells to mitigate organ damage will require the ability to monitor teratoma formation with high sensitivity and specificity [18].…”

Human umbilical cord blood derived mesenchymal stem cells were differentiated into pancreatic endocrine cell by Pdx-1 electrotransfer

“…ESCs can be differentiated into all cell type originated from three germ layers of the embryo, including beta cells. However, ESC based applications were barricaded by ethical reasons and the high rate of malignant tumor formation (Abdelalim et al, 2014;Su et al, 2011). MSCs possess the ability of high self-renewal, easily develop into various cells, especially modulate immune system; so they were especially interested in diabetes mellitus type 1 treatment (Helledie et al, 2008;Wu and Mahato, 2014).…”