52. Gut Enriched Kruppel-Like Factor (GKLF) Inhibits Differentiation and Induces Self-Renewal of Murine Embryonic Stem Cells

Abstract

One of the challenges of gene therapy techniques, in addition to the adequate delivery and expression of the desired gene, is the maintenance of stem cell self-renewal during ex vivo manipulations. However, the genes needed for stem cell self-renewal are largely unknown. Homozygous mutant (Shp-2Δ46-110) embryonic stem (ES) cells bearing a targeted exon 3 deletion of the Shp-2 protein tyrosine phosphatase gene exhibit increased sensitivity to Leukemia Inhibitory Factor (LIF) and increased self-renewal potential as assayed by 2° embryoid body frequency upon secondary plating. LIF maintains the pluripotency of ES cells, suggesting that molecules stimulated by LIF are critical for self-renewal. Based on the functional characteristics of the Shp-2Δ46-110 ES cells, we hypothesized that LIF-responsive genes in these cells would be good candidates for molecules vital for stem cell self-renewal. Using a microarray analysis approach, we generated and previously reported a list of LIF-induced genes in Shp-2Δ46-110 ES cells (International Society of Experimental Hematology, 2002, Abstract #326). The two most significantly upregulated genes were Suppressor of Cytokine Signaling-3 (SOCS-3) and Gut Enriched Kruppel-Like Factor (GKLF). In order to assess the capacity of these proteins to affect stem cell self-renewal, each cDNA was introduced into ES cells followed by examination of ES cell differentiation and self-renewal. The FLAG-tagged SOCS-3 and GKLF cDNAs were subcloned into the murine retroviral vector, pMIEG3, in tandem with the enhanced green fluorescent protein (EGFP) cDNA. WT R1 ES cells were transduced and EGFP-positive cells were selected using FACS. Selected cells were subjected to differentiation followed by secondary assays for hemangioblasts, primitive erythroid colonies, and 2° embryoid bodies. We found that ES cells expressing exogenous SOCS-3 (compared to ES cells tranduced with vector alone) had an increased capacity to differentiate to both hemangioblasts (17+/−2.5 v. 6+/−2.4, p = 0.02, student's t test) and to primitive erythroid cells (100+/−5.4 v. 47.6 +/−20.1, p = 0.03, student's t test). There was no difference in the number of 2° embryoid bodies (6.9+/−1.3 v. 5.5+/−0.7, p>0.5). However, in contrast, introduction of GKLF inhibited the differentiation capacity of the ES cells as we observed a trend toward lower numbers of primitive erythroid colonies compared to vector alone (10.6+/−2.1 v. 47.6+/−20.1, p = 0.09, student's t test). Likewise, ES cells expressing exogenous GKLF had a significantly greater capacity to undergo self-renewal as assessed by 2° embryoid body formation compared to cells transduced with vector alone (117.3+/−19.3 v. 5.5+/−0.8, p = 0.0002, student's t test). In both studies, we demonstrated that the exogenously FLAG-tagged molecules were expressed during ES cell differentiation. GKLF is highly expressed in the epithelial crypts of the colon, the site of somatic epithelial stem cells. This previous information along with our data qualifies GKLF as a reasonable candidate for a protein necessary for somatic stem cell self-renewal. We are currently transducing GKLF into hematopoietic stem cells (HSC) followed by competitive transplant assays to determine if GKLF has the capacity to enhance HSC self-renewal.