Adipose tissue derived mesenchymal stem cells AT MSC are

Adipose tissue derived mesenchymal stem order Cycloheximide (AT-MSC) are an easily available autologous source of multipotent cells for cell therapy applications (Pittenger et al., 1999; Zuk et al., 2002). Like HSC, MSC derive from embryonic mesoderm. When cultured in hematopoietic differentiation medium, AT-MSC could be shown to adopt gene expression and functionality similar to macrophages of the hematopoietic lineage (Freisinger et al., 2010). In 2006, Yamanaka and his colleagues showed how transduction of a set of defined genes could reprogram fibroblasts to become pluripotent cells (Takahashi and Yamanaka, 2006; Takahashi et al., 2007; Park et al., 2008). Recently, reprogramming to pluripotency has been achieved by single gene transduction of OCT4 (Kim et al., 2009). In another study, OCT4 transduction of human fibroblasts was shown to generate hematopoietic progenitor cells (Szabo et al., 2010). Based on these studies, and starting with MSC that have a certain similarity to HSC, we hoped to be able to generate HSC by transduction of a single gene, and at the same time avoid the potential dangers of pluripotency that may follow OCT4 transduction. Studies in zebrafish have identified the caudal-related homeobox containing transcription factors (cdx) and posterior hox genes as key regulators for blood formation during embryonic development. By the activation of downstream hox targets, cdx genes pattern hematopoiesis in zebrafish (Davidson et al., 2003; Davidson and Zon, 2006) and can promote hematopoiesis from mouse embryonic stem cells (mESC) (Wang et al., 2005b; Lengerke et al., 2007, 2008). The ectopic expression of Cdx4 and HoxB4 genes enables the derivation of mESC‐derived HSC (Wang et al., 2005b). Furthermore, bone morphogenetic protein 4 (BMP4), Wnt3a and hematopoietic cytokines direct blood formation via activation of Cdx and Hox genes in mESC and in human induced pluripotent stem cells (iPS) (Lengerke et al., 2008, 2009) suggesting the conservation of blood patterning pathways between mouse and human hematopoietic development. Therefore, we assumed that the CDX–HOX pathway might play a similarly central role in human hematopoietic development, with CDX4 acting as a master switch gene on downstream HOX genes, which in turn would induce the genetic program required for hematopoiesis. CDX4 is not expressed in AT-MSC. In order to directly reprogram AT-MSC to HSC, we used a retroviral transduction system to induce stable human CDX4 expression in these cells. Additionally, we exposed the cells to the hematopoietic cytokines interleukin-3 (IL3), stem cell factor (SCF), Fms-related tyrosine kinase-3 (Flt-3) ligand and thrombopoietin (TPO), or to BMP4, or to the epigenetic modifiers 5-azacytidine (5-AzaC) and trichostatin A (TSA). However, despite high expression of CDX4 at the mRNA and protein levels, real-time PCR results showed no effect on downstream HOX genes or other genes of importance for hematopoietic development, and functional colony forming assays showed no hematopoietic colonies. These results indicate that the introduction of a single master switch gene and exposure of the AT-MSC to epigenetic modifiers or hematopoietic cytokines is not sufficient to induce cell fate changes in MSC, at least under these conditions.
Materials and methods
Results
Discussion MSC have the potential to differentiate into cells and organs of mesodermal origin, such as bone, cartilage, fat, tendons and skeletal muscle (Pittenger et al., 1999; Barry and Murphy, 2004). There is also increasing evidence for their ability to transdifferentiate into cells of other lineages (Schaffler and Buchler, 2007). For this reason we assumed that it might be possible to reprogram MSC to become HSC, which are also derived from mesoderm. Primitive haematopoiesis is initiated in the yolk sac blood islands derived from the posterior mesoderm by the formation of embryonic erythroid cells and endothelial cells (Palis et al., 1995; Palis and Yoder, 2001; Ferkowicz and Yoder, 2005). In zebrafish, cdx4 mutants fail to specify blood progenitors, while cdx4 overexpression during development induces blood formation (Davidson et al., 2003). In this study, the blood deficiency induced by the cdx4 mutation could be rescued by the overexpression of several hox genes. These findings suggested that, in zebrafish, cdx4 regulates hox genes, and that this axis is essential for the specification of hematopoiesis during embryogenesis. Later, the Cdx-Hox pathway was found to be involved also in murine hematopoiesis (Wang et al., 2005b; Lengerke et al., 2008). Here, the coordinated activity of Bmp4 and Wnt signaling was found to be important for hematopoietic fate specification. In mESC the ectopic expression of Cdx4 was found to promote hematopoietic progenitor formation (Wang et al., 2005b). Thus, in zebrafish and mice, the Cdx4/Hox axis seems to be essential for embryonic hematopoiesis, and ectopic overexpression of members of this pathway on a background of pluripotency will promote hematopoietic development. In human cells, the role of these genes is not so clear. Overexpression of HOXB4 was found to promote hematopoietic development in human ESC (Bowles et al., 2006). However, hematopoietic repopulating cells have also been generated from hESC independent of ectopic HOXB4 expression where HOXB4, as a single master switch gene was unable to induce hematopoietic repopulating capacity from hESC (Wang et al., 2005a). To the best of our knowledge, no studies have been able to demonstrate that the CDX4/HOX axis is of the same importance for the specification of hematopoiesis in the human system as that which has been clearly shown in mice and zebrafish.