SECTOR: Vivimos El Tarra institucional y administrativamente

Regarding embryonic stem cells (ESCs) or ESC-derivatives, the cells were injected into developing metanephroi, similarly as for kidney progenitors and AFSCs, but were found primarily in tubular compartments (Kim and Dressler 2005; Steenhard et al. 2005; Vigneau et al. 2007) (Table 1.1). The injection of undifferentiated ESCs into E13 mouse kidney rudiments resulted after 5 days of culture, in the formation of large tubule-like structures consisting of cells displaying apical microvilli, junctional complexes and basal bodies, surrounded by a basement membrane. Furthermore, such ESC-derived structures stained with LTA, a marker for proximal tubules. It has been also observed that ESCs did not mix with native kidney cells to create chimeric tubules in the metanephroi. In addition, injected ESCs were rarely observed in developing glomeruli (Steenhard et al. 2005). Other groups injected pre-differentiated ESCs into kidney rudiments (Kim and Dressler 2005; Vigneau et al. 2007). For instance, Kim and Dressler used undifferentiated ESCs to form embryoid bodies (EBs), which after 5 days of culture, were further induced with retinoic acid, activin-A and bone morphogenic protein-7. Following the induction, the cells started to express genes involved in early kidney development, such as Pax2, Wt1, Wnt4, Lim1, Six2, Eya1 and Gdnf. These differentiated ESCs engrafted into tubules of E12.5 mouse kidney rudiments after 5 days of in vitro culture. Furthermore, tubules generated by the differentiated cells stained with laminin and LTA. In addition, EB-derived cells that were not induced with the nephrogenic cocktail had a lower ability to incorporate into tubules and were found mainly in non-tubular structures (Kim and Dressler 2005). Another group demonstrated that if ESCs were differentiated towards an early renal phenotype prior to their injection into the

E11.5 kidney rudiment, the cells were found in pretubular aggregates after 4 days of culture. Accordingly, when injected into the kidneys of newborn mice, the cells incorporated into proximal tubules. The cells used in these experiments were derived from EBs, similarly as in the previous study, but with the difference that they were cultured in the presence of activin-A for 4 days before injection. This resulted in high expression levels of the nascent mesodermal marker, brachyury, which was subsequently used to divide the EB cell population in two separate fractions. Interestingly, in comparison to the brachyury positive fraction, which integrated into proximal tubules, the brachyury negative cells integrated into ureteric buds (Vigneau et al. 2007). Recently, it has been shown that conditioned medium from a UB culture can trigger ESCs previously induced by retinoic acid and activin, to differentiate towards a renal phenotype, as assessed by expression of WT1, Pax2 and binding of Dolichos biflorus agglutinin (Ren et al. 2010).

Table 1.1 Different techniques used to introduce stem cell/progenitor populations into the embryonic kidney environment with the aim of differentiating them towards a kidney-specific phenotype.

Stem cell/progenitor

No. of cells

Outcome Methodology Reference

Human MSCs 1000 Glomerular and

tubular epithelium and interstitium

Injection into intermediate mesoderm of a rat embryo

or E13 rat kidney

Yokoo et al. 2005; Yokoo

et al. 2006

Human MSCs 50-100 Wolffian duct Injection into intermediate

mesoderm of a chicken embryo

Fukui et al. 2009

Human AFSCs 1000 Stroma, renal

vesicle, C- and S- shaped bodies

Injection into E12.5-E18 mouse kidney rudiment

Perin et al. 2007

Human AFSCs 10 000 Developing

nephron, UB

Recombination of AFSCs with E11.5 mouse kidney

cells (1:10) Siegel et al. 2010 Human kidney progenitors 4500 Integration into tubules

Injection into E12.5 mouse kidney rudiment

Ward et al. 2011

Mouse ESCs 1500 Proximal tubules Injection into E12-E13

mouse kidney rudiment

Steenhard et al. 2005 Mouse ESC- derived progenitors 1000- 2000

Proximal tubules Injection into E12.5 mouse

kidney rudiment Kim and Dressler 2005 Mouse ESC- derived progenitors 300 Pretubular aggregates

Injection into E11.5 mouse kidney rudiment

Vigneau et al. 2007 Mouse kidney

progenitors

100 Ureteric buds and

condensing metanephric mesenchyme

Injection into E12.5 mouse kidney rudiment Challen et al. 2006 Rat kidney progenitors 200 Interstitium, ureteric buds, proximal tubules

Injection into E15 rat kidney rudiment

Maeshima et al. 2006

Aim of the study

The aim of the study is to investigate the potential of mesenchymal stem cells (MSCs), a multipotent cell population (Prockop 1997), to contribute to in vitro nephrogenesis by differentiating into kidney-specific cell types in a metanephric environment of mouse kidney rudiment. MSCs might be potential candidates for stem cell-based renal tissue generation as they not only exert renoprotective effects in different acute kidney injury models, but also engraft into the tubules of injured kidneys, and to some extent, differentiate into tubular epithelial cells (Herrera et al. 2004; Morigi et al. 2004; Qian et al. 2008; Li et al. 2010). Moreover, human MSCs were shown to give rise to nephron-specific cell types after they underwent specific reprogramming in the embryonic environment of the metanephric kidney (Yokoo et al. 2005; Yokoo et al. 2006). Additionally, human MSCs were shown to ameliorate renal pathologies when incorporated into embryonic kidneys containing renal defects (Yokoo et al. 2005; Guillot et al. 2008). Nevertheless to achieve a significant contribution of MSCs to metanephric development, a sophisticated methodology is required, as demonstrated by Yokoo et al. (Yokoo et al. 2005). Since the direct injection of human MSCs into the developing kidney in vitro, is insufficient to enable MSC differentiation, the cells need to be genetically modified to over-express glial cell- derived neurotrophic factor before they are injected into the intermediate mesoderm of an embryo, followed by ex utero culture of the embryos (Yokoo et al. 2005; Yokoo et al. 2006; Fukui et al. 2009). The current study is therefore aimed at re-evaluating the renogenic capacity of mouse and human bone marrow-derived MSCs using a much simpler protocol described by Unbekandt and Davies, which is based on disaggregation and subsequent re-aggregation of mouse metanephroi (Unbekandt and Davies 2010). This new methodology allows incorporation

of cells from different origins into kidney rudiments to form kidney chimeras and to determine the contribution of the cells to nephrogenesis in an in vitro environment, which mimics metanephric development. Furthermore, in this study an attempt will be made to enhance the potential of MSCs to engraft into structures of developing kidney chimeras by incubating the cells with conditioned medium from a kidney cell culture. The use of conditioned medium to induce differentiation of MSCs has been described before. It has been demonstrated that MSCs can adopt the characteristics of the cells from which the conditioned medium was derived (Rivera et al. 2006; Pan et al. 2008; Baer et al. 2009; Schittini et al. 2010). The conditioned medium from neonatal kidney cells will be used in this study to pre-condition both mouse and human MSCs and accordingly the renogenic potential of pre-conditioned MSC will be evaluated. Finally, the paracrine activity of MSCs was shown to play a major role in promoting recovery from kidney injury (Togel et al. 2005; Bi et al. 2007; Imberti et al. 2007; Semedo et al. 2007; Togel et al. 2007; Togel et al. 2009). However, it is not clear what effect factors secreted by MSCs might have on embryonic kidneys. Therefore the paracrine action of MSCs on metanephric kidneys will be additionally assessed in this study.

The following is a summary of the most important issues addressed in this study:

• the ability of the MSC to contribute to developing renal structures in a chimeric kidney culture assay based on the protocol of Unbekandt and Davies

• the potential of other cells, such as embryonic stem cells and neonatal kidney cells, to contribute to developing renal structures using the protocol of Unbekandt and Davies

neonatal kidney cells using the protocol of Unbekandt and Davies