High-Efficiency Stem Cell Fusion-Mediated Assay Reveals Sall4 as an Enhancer of Reprogramming

Baylor College of Medicine, United States of America
PLoS ONE (Impact Factor: 3.23). 02/2008; 3(4):e1955. DOI: 10.1371/journal.pone.0001955
Source: PubMed


Several methods allow reprogramming of differentiated somatic cells to embryonic stem cell-like cells. However, the process of reprogramming remains inefficient and the underlying molecular mechanisms are poorly understood. Here, we report the optimization of somatic cell fusion with embryonic stem cells in order to provide an efficient, quantitative assay to screen for factors that facilitate reprogramming. Following optimization, we achieved a reprogramming efficiency 15-590 fold higher than previous protocols. This allowed observation of cellular events during the reprogramming process. Moreover, we demonstrate that overexpression of the Spalt transcription factor, Sall4, which was previously identified as a regulator of embryonic stem cell pluripotency and early mouse development, can enhance reprogramming. The reprogramming activity of Sall4 is independent of an N-terminal domain implicated in recruiting the nucleosome remodeling and deacetylase corepressor complex, a global transcriptional repressor. These results indicate that improvements in reprogramming assays, including fusion assays, may allow the systematic identification and molecular characterization of enhancers of somatic cell reprogramming.

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    • "Supporting a role of SALL4 in pluripotency, this factor enhanced reprogramming of human and mouse somatic cells to induced pluripotent stem cells [Tsubooka et al., 2009]. In another experimental setup, where somatic cells were fused with ES cells to regain pluripotency, SALL4 also enhanced reprogramming [Wong et al., 2008]. Interestingly, besides pluripotent stem cells, SALL4 has been implicated in multipotent hematopoietic stem cell and fetal hepatic progenitor cell function [Ma et al., 2006; Yang et al., 2007, 2008b; Oikawa et al., 2009]. "
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    ABSTRACT: SALL4 (sal-like protein 4) is a pluripotency transcription factor, which is highly expressed in embryonic stem (ES) cells and which is essential for mouse preimplantation development. In adult mouse organs, Sall4 mRNA is highly expressed in the testis and ovary, while there is only little or no expression in other organs. There is also a high expression of SALL4 in human testicular germ cell tumors. However, there is as yet no detailed analysis of SALL4 expression during mammalian testicular development. We analyzed SALL4 expression in ES cells, preimplantation embryos, and the developing and adult testis of a nonhuman primate (NHP) species, the common marmoset monkey (Callithrix jacchus). Immunofluorescence revealed SALL4 in the nuclei of marmoset ES cells and preimplantation embryos. Marmoset SALL4 isoform analysis in ES cells and newborn and adult testis by RT- PCR and Western blotting showed two different isoforms, SALL4-A and SALL4-B. Immunohistochemistry localized this transcription factor to the nuclei of primordial germ cells and most gonocytes in the prenatal and early postnatal marmoset testis. In the pubertal and adult testis SALL4 was present in undifferentiated spermatogonia. In the developing and adult human and mouse testis SALL4 expression mimicked the pattern in the marmoset. Adult testes from additional NHP species, the treeshrew, the cat and the dog also exhibited SALL4 in undifferentiated spermatogonia, indicating a conserved expression in the mammalian testis. Taking into account the importance of SALL4 for mouse development, we conclude that SALL4 may play an important role during mammalian germ cell development and is involved in the regulation of spermatogonial proliferation in the adult testis.
    Cells Tissues Organs 05/2012; 196(3):206-20. DOI:10.1159/000335031 · 2.14 Impact Factor
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    • "Sall4 is a transcription factor, sharing several cobound sites with Oct4, Sox2, and Nanog (Lim et al. 2008; Yang et al. 2008), that could also improve the efficiency of the somatic cell reprogramming in MEFs. This was demonstrated by different approaches of cell fusion and direct reprogramming, using defined factors (Tsubooka et al. 2009; Wong et al. 2008). However, the effects of Sall4 on human somatic cells (e.g., human dermal fibroblasts) have been proven to be different . "
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    ABSTRACT: Induced pluripotent stem cells are generated by direct reprogramming of somatic cells with the introduction of defined transcription factors or other means. Clinical applications of induced pluripotent stem cells are the latest of stem cell therapy approaches due to overcoming problems associated with insufficient cells from conventional sources and immune rejections. In practice, this is restricted by 4 major barriers including the use of genetic manipulations for delivering the reprogramming factors, low efficiency of this process, slow kinetics of the direct reprogramming, and potential for tumor development. Here, we review the latest achievements in improving reprogramming efficiency by alternative strategies. These alternatives mainly involve the replacement of genetic reprogramming factors with small molecules or other factors.
    Biochemistry and Cell Biology 12/2011; 90(2):115-23. DOI:10.1139/o11-064 · 2.15 Impact Factor
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    • "Expression analysis and functional studies of the short and long forms of Xenopus Sall4 during limb regeneration at the regeneration-complete and -incomplete state has the potential to provide insight into dedifferentiation/reprogramming. SALL4 is an enhancer (positive regulator ) of differentiated cell reprogramming toward an embryonic stem cell–like state and the reprogramming activity of SALL4 is associated with its transcription repressor activity located in the N-terminal domain of the protein (Wong et al., 2008). "
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    ABSTRACT: A central feature of epimorphic regeneration during amphibian limb regeneration is cellular dedifferentiation. Two questions are discussed. First, what is the origin and nature of the soluble factors involved in triggering local cellular and tissue dedifferentiation? Secondly, what role does the key stem cell transcription factor Sall4 play in reprogramming gene expression during dedifferentiation? The pattern of Sall4 expression during Xenopus hindlimb regeneration is consistent with the hypothesis that Sall4 plays a role in dedifferentiation (reprogramming) and in maintaining limb blastema cells in an undifferentiated state. Sall4 is involved in maintenance of ESC pluripotency, is a major repressor of differentiation, plays a major role in reprogramming differentiated cells into iPSCs, and is a component of the stemness regulatory circuit of pluripotent ESCs and iPSCs. These functions suggest Sall4 as an excellent candidate to regulate reprogramming events that produce and maintain dedifferentiated blastema cells required for epimorphic regeneration.
    Developmental Dynamics 05/2011; 240(5):979-89. DOI:10.1002/dvdy.22554 · 2.38 Impact Factor
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