A Scalable Approach to Prevent Teratoma Formation of Human Embryonic Stem Cells

From the Division of Biological Sciences, University of California at San Diego, La Jolla, California 92093-0322 and.
Journal of Biological Chemistry (Impact Factor: 4.57). 08/2012; 287(39):32338-45. DOI: 10.1074/jbc.M112.383810
Source: PubMed


As the renewable source of all cell types in the body, human embryonic stem cells (hESCs) hold great promise for human cell therapy. However, one major bottleneck that hinders the clinic application of hESCs is that hESCs remaining with their differentiated derivatives pose cancer risk by forming teratomas after transplantation. NANOG is a critical pluripotency factor specifically expressed in hESCs but rarely in their differentiated derivatives. By introducing a hyperactive variant of herpes simplex virus thymidine kinase gene into the 3'-untranslated region of the endogenous NANOG gene of hESCs through homologous recombination, we developed a safe and highly scalable approach to efficiently eliminate the teratoma risk associated with hESCs without apparent negative impact on their differentiated cell types. As thymidine kinase is widely used in human gene therapy trials and is the therapeutic target of U. S. Food and Drug Administration-approved drugs, our strategy could be effectively applied to the clinic development of hESC-based human cell therapy.

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    • "Various strategies developed to reduce this risk of teratoma formation include prolonged pre-differentiation of ESCs in vitro, blocking signaling pathways that promote proliferation, induction of apoptosis of proliferative ESCs, sorting cells expressing precursor markers, and deleting undifferentiated ESCs immunologically, genetically, and chemically (7–18). However, it is difficult to obtain a yield of 100% pure differentiated stem cells for transplantation: the contamination of grafts with undifferentiated cells can give rise to teratoma formation (19–21). Furthermore, teratoma could potentially develop into highly malignant teratocarcinoma, which constitutes of persistent and undifferentiated stem cells (22). "
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    ABSTRACT: Stem cell therapies have had tremendous potential application for many diseases in recent years. However, the tumorigenic properties of stem cells restrict their potential clinical application; therefore, strategies for reducing the tumorigenic potential of stem cells must be established prior to transplantation. We have demonstrated that syngeneic transplantation of embryonic stem cells (ESCs) provokes an inflammatory response that involves the rapid recruitment of bone marrow-derived macrophages (BMDMs). ESCs are able to prevent mature macrophages from macrophage colony-stimulating factor (M-CSF) withdrawal-induced apoptosis, and thus prolong macrophage lifespan significantly by blocking various apoptotic pathways in an M-CSF-independent manner. ESCs express and secrete IL-34, which may be responsible for ESC-promoted macrophage survival. This anti-apoptotic effect of ESCs involves activation of extracellular signal-regulated kinase (ERK)1/2 and PI3K/Akt pathways and thus, inhibition of ERK1/2 and PI3K/AKT activation decreases ESC-induced macrophage survival. Functionally, ESC-treated macrophages also showed a higher level of phagocytic activity. ESCs further serve to polarize BMDMs into M2-like macrophages that exhibit most tumor-associated macrophage phenotypic and functional features. ESC-educated macrophages produce high levels of arginase-1, Tie-2, and TNF-α, which participate in angiogenesis and contribute to teratoma progression. Our study suggests that induction of M2-like macrophage activation is an important mechanism for teratoma development. Strategies targeting macrophages to inhibit teratoma development would increase the safety of ESC-based therapies, inasmuch as the depletion of macrophages completely inhibits ESC-induced angiogenesis and teratoma development.
    Frontiers in Immunology 07/2014; 5:275. DOI:10.3389/fimmu.2014.00275
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    • "The flow cytometric analysis of the surface expression of hESC-specific markers, TRA 1-61, TRA 1-81, SSEA3, and SSEA4, was analyzed as described elsewhere (Rong et al., 2012). Briefly, 5 3 10 5 cells were washed with PBS and stained for 1 hr at room temperature with primary antibody. "
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    ABSTRACT: Human embryonic stem cells (hESCs) hold great promise for cell therapy as a source of diverse differentiated cell types. One key bottleneck to realizing such potential is allogenic immune rejection of hESC-derived cells by recipients. Here, we optimized humanized mice (Hu-mice) reconstituted with a functional human immune system that mounts a vigorous rejection of hESCs and their derivatives. We established knockin hESCs that constitutively express CTLA4-Ig and PD-L1 before and after differentiation, denoted CP hESCs. We then demonstrated that allogenic CP hESC-derived teratomas, fibroblasts, and cardiomyocytes are immune protected in Hu-mice, while cells derived from parental hESCs are effectively rejected. Expression of both CTLA4-Ig, which disrupts T cell costimulatory pathways, and PD-L1, which activates T cell inhibitory pathway, is required to confer immune protection, as neither was sufficient on their own. These findings are instrumental for developing a strategy to protect hESC-derived cells from allogenic immune responses without requiring systemic immune suppression.
    Cell stem cell 01/2014; 14(1):121-30. DOI:10.1016/j.stem.2013.11.014 · 22.27 Impact Factor
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    • "The GCV treatment has to be applied directly after ES cell injection to prevent tumor formation caused by pluripotent cells as it is not able to eliminate or stop the growth of established tumors. This in accordance with previously published results [49]. "
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    ABSTRACT: Embryonic stem cells (ES) and induced pluripotent stem (iPS) cells represent promising tools for cell-based therapies and regenerative medicine. Nevertheless, implantation of ES cell derived differentiated cells holds the risk of teratoma formation due to residual undifferentiated cells. In order to tackle this problem, we used pluripotent stem cells consisting of ES and iPS cells of mouse genetically modified by lentiviral vectors (LVs) carrying herpes simplex virus thymidine kinase (HSV-TK) under the control of different promoters of pluripotency genes. Cells expressing TK in turn are eliminated upon administration of the prodrug ganciclovir (GCV). Our aim was to study the conditions required for a safe mechanism to clear residual undifferentiated cells but using low MOIs of lentiviruses to reduce the risk of insertional mutagenesis. Our in vitro data demonstrated that TK expression in pluripotent stem cells upon treatment with GCV led to elimination of undifferentiated cells. However, introduction of hygromycin resistance in the LV transduced ES cells followed by pre-selection with hygromycin and GCV treatment was required to abolish undifferentiated cells. Most importantly, transplantation of pre-selected ES cells that had been transduced with low MOI LV in mice resulted in no teratoma development after GCV treatment in vivo. Taken together, our data show that pre-selection of ES cells prior to in vivo application is necessary if vector integration events are minimized. The study presented here gives rise to safer use of pluripotent stem cells as promising cell sources in regenerative medicine in the future.
    PLoS ONE 07/2013; 8(7):e70543. DOI:10.1371/journal.pone.0070543 · 3.23 Impact Factor
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