In 1981, ES cells were isolated for the first time from mice. This major break through, revolutionized the field of developmental biology (Martin, 1981
). ES cells can undergo prolonged self-renewal and differentiation, thus providing a tool to investigate the molecular mechanisms occurring during differentiation from the embryonic stage to the adult phenotype. These cells are considered to be pluripotent and can differentiate into almost all cells that arise from the three embryonic germ layers (Alison et al., 2002
). In addition, ES cells can differentiate into multiple embryonic and adult cell type’s in-vitro, but rarely cells of endodermal linage (Trounson, 2006
). Differentiation in an in-vivo environment yields the full developmental potential of undifferentiated ES cell lines. For example, ES cells injected into severe combined immunodeficient (SCID) mice formed benign teratomas (Bradley et al., 1984
; Gertow et al., 2004
; Heins et al., 2004
; Przyborski et al., 2004
; Reubinoff et al., 2000
; Thomson et al., 1998
). In the ES cell teratomas, advanced differentiated structures from all three embryonic germ cell (EG) layers were present, including smooth muscle, bone, cartilage, gut, respiratory epithelium, keratinizing squamous epithelium, hair, neural epithelium, and ganglia.
The presence of stem and/or progenitor cells within the bladder is unknown, but hypothetically should exist. To identify stem cells within the vast population of cells in the bladder would be challenging, especially without bladder specific stem/progenitor cell markers. In attempt to identify bladder stem cells, we forced ES cells to differentiate into bladder structures rather than a teratoma. Thus, we document a transition of the ES cells to early endoderm and then to the progenitor cells that give rise to bladder stem cells and thence urothelium. Rather than isolating a stem cell, we were watching it develop.
We hypothesized that if we can harness ES cells to produce other organs rather than teratomas, we will have a unique opportunity to investigate the molecular pathways that determine the stem cells for multiple organs during embryonic development. In order to study these pathways, it is important to achieve almost 100′ conversion of the ES cell to only one tissue type. Recently Taylor et al. (Taylor et al., 2006
) reported that human embryonic stem cells recombined with rat urogenital mesenchyme (UGM) gave rise to immature and mature human prostate tissue, supporting the hypothesis that specific organ tissues can be influenced by inductive mesenchyme to form from ES cells. Although, the precise inductive events regulating embryonic pattern formation are still unknown; it is known that UGM isolated from embryonic day 18 urogenital sinus can induce freshly isolated adult bladder urothelium to develop a prostate (Aboseif et al., 1999
; Donjacour and Cunha, 1993
; Neubauer et al., 1983
) and that the UGM is required for prostate development (Cunha, 1972
). Although this inductive nature of UGM is well established, it is unknown whether the bladder urothelium undergoes a trans-differentiation to prostatic epithelium or if bladder and prostate share a common stem cell that is controlled by different inductive mesenchyme.
Initial experiments showed that no significant growth and differentiation into teratomas occurred at less than 500 ES cells per graft. At 1000 cells per graft, complex structures representing a teratoma occurred. Staining for a urothelial marker, uroplakin, confirmed the lack of bladder tissues spontaneously developing. Although theoretically ES cells could generate urothelium and bladder tissues within a teratoma, it was important to determine the minimum number of ES cells needed to form a teratoma with the goal of redirecting these ES cells to form only one organ type.
Based upon the findings that 1000 ES cells + 1 EBLM shell per graft yielded small foci of uroplakin expressing bladder tissue within a teratoma, we hypothesized that the system could be pushed further to form only pure bladder tissue devoid of teratomatous elements by simply increasing the ratio of EBLM to ES cells. We empirically increased both the number of EBLM shells from one to four in each graft, along with optimizing the number of ES cells from 1000 to 1500. This ratio proved to be successful, the recombinants no longer exhibited teratoma structures, only pure uroplakin expressing urothelium with mature bladder tissues. Amazingly, the ES cells conversion into mature urothelium exhibited cellular organization that was complex such that the basilar located cells were p63 positive and the central luminal urothelium remained p63 negative. Within the recombinant stromal compartment, smooth muscle layers correctly differentiated and neuronal cells organized to form ganglionic tissue. Additional neuronal cells were also present interlaced among smooth muscle fibrils. These neuronal elements most likely originated from the ES cells but we cannot rule out the host kidney as the source. Clearly, the neuronal cells did not develop from the rat EBLM since we confirmed the mouse species specificity of these cells with the NeuN antibody. No other reports have shown that neuronal elements would develop in bladder tissue following a recombination of bladder urothelium with bladder mesenchyme. Remarkably, the conversion of ES cells to the bladder phenotype exhibited all the cellular organization required for a functional bladder.
Embryonic bladders can only be readily identified around 14 days of gestation in the mouse. Therefore the earlier events that orchestrate endodermal lineage differentiation have been impossible to investigate. Each stage that occurs in the recombinant tissues reflects stages of ES cells conversion into endoderm, then from endoderm to bladder stem cells, then from stem cells to mature urothelium within bladder tissue. Further studies will be needed to follow their complete developmental progression.
No studies or models to date have described endodermal lineage protein expression patterns in the developing bladder to adult maturation. In our time point experiments, we first detected the endodermal markers of Foxa1 and Foxa2 at 7 days, then extensively by 14 days. During these stages, uroplakin was undetectable, consistent with studies that have shown that immature urothelium does not express uroplakin (Lavelle et al., 2002
; Romih et al., 1998
). Although these cells were endodermal and looked destined to become urothelium, they were not mature urothelium at these two early time points.
Since little is known about the gene expression patterns in the developing bladder, we based our experimental parameters on what is known in the prostate since both organs are hindgut derivatives. Mirosevich et al. (Mirosevich et al., 2005
) reported that embryonic day 18 rodent prostate and bladders express Foxa1 in the epithelial compartments and Besnard et al. (Besnard et al., 2004
) identified Foxa1 expression in the urothelium of adult bladders. It is known that Foxa2 expression in the developing prostate occurs in very early epithelial budding during embryogenesis and that its expression coincides with Foxa1 expression (Mirosevich et al., 2005
). As the developing prostate forms more mature glands, Foxa1 expression is retained in the epithelium through adulthood and Foxa2 is extinguished post-natally. Our findings parallel those in the prostate, Foxa1 and Foxa2 expression were detected early in the 7 and 14 day recombinants, but by 42 days only Foxa1 remained and Foxa2 was almost undetectable. We also performed IHC with antibodies to both Fox proteins in native E16, postnatal day 1, and adult mouse bladders. In E16 tissues, Foxa1 was expressed in all urothelium with Foxa2 expression limited to only a few cells. In postnatal day 1 and adult bladder tissues, Foxa1 was again expressed in all urothelium but Foxa2 expression was completely undetectable. These findings illustrate a temporal relationship of Foxa protein expression patterns that have not been previously reported in the bladder and it demonstrates the potential power to use this model not only in studying bladder development but in other organ systems as well. These findings capture a time period during development illustrating that Foxa2 is highly expressed early in bladder progenitor cells, then completely turns off in mature urothelium. The exact role of the Fox proteins in bladder development remains unclear, although we have demonstrated a novel model in which to characterize endodermal related events involved in bladder embryogenesis.
Overall, developmental models are lacking in which to study embryonic differentiation into a specific organ. We have shown that mouse ES cells, in the appropriate inductive signaling environment, can undergo endodermal lineage transformation into mature urothelium. Our model yields a powerful example of the temporal and spatial events occurring during endodermal differentiation of the bladder. These findings suggest that ES cells can be controlled to differentiate into specific organs opening up the opportunity to characterize the molecular and cellular events that give rise to organ stem cells.