The mammalian metanephric kidney develops from the intermediate mesoderm by a series of reciprocal inductive interactions between the metanephric blastema and the ureteric bud (Dressler, 2006
). Signals from the metanephric blastema drive branching morphogenesis of the ureteric bud which, in turn, induces aggregation and differentiation of mesenchymal cells or the blastema surrounding the bud tips. The renal vesicle is the first recognizable structure to form in the differentiating mesenchyme, followed by development of S- and C-shaped bodies as the vesicle matures. Endothelial cell migration into the cleft of the S-shaped body results in development of glomerular tuft capillaries and the ensuing interactions between endothelial cells and surrounding mesenchymal-derived epithelium results in further maturation of the renal filtration unit, the glomerular basement membrane.
PAX2 and Vimentin immunoreactivity shown in developing rhesus monkeys during kidney ontogeny is similar to findings in human specimens at similar gestational ages (Winyard, et al., 1996
, Naruse et al., 2000
) and further supports the importance of the monkey as a model for human nephrogenesis (Lee et al., 2001
; Rodriguez et al., 1996
). Similar to findings in humans and rodents, PAX2 is an important marker of kidney development in the monkey with strong expression at the ureteric bud tips, the condensed mesenchyme and renal vesicle, and in the developing visceral and parietal layers of the capsule. Intense PAX2 staining was also notable in late gestation medullary regions and may be consistent with proposed progenitor cell populations in the renal papilla (Al-Awqati and Oliver, 2006
; Oliver et al., 2004
). In the developing glomerulus, Vimentin expression was initially noted in the early glomerular tufts and with maturation was shown additionally in the visceral epithelium in mature glomeruli, in accordance with previous reports in humans (Naruse et al., 2000
; Holthöfer et al., 1984
This complex and intricate process of kidney development provides a logical blueprint for in vitro
production of precursors from hESC that may be useful for kidney repair. The panel of markers used in these studies includes genes known to be expressed in the intermediate mesoderm, early kidney precursors, differentiating medullary and cortical regions, and more mature kidney cell populations of the collecting system and excretory component. The expression pattern of spontaneously differentiating hEBs showed greater expression of genes important in early kidney ontogeny when compared to those genes indicative of more mature kidney cell types, which was not unexpected (). When RA7 was added to the culture medium, hEBs differentiated rapidly over time as evident by declining levels of OCT4 and NANOG expression ( and ) in both suspension and laminin-substrate culture systems. These findings were not observed in gelatin-substrate monolayer culture where OCT4 expression remained fairly constant over time while NANOG expression increased 2-fold () despite strong upregulation of intermediate mesodermal markers SIX2, WT1, and OSR1. Although additional characterization is needed to determine the exact nature of these cells, the results are suggestive of a population of renal precursors that express OCT4 together with renal differentiation markers, similar to the multipotent renal progenitor cells characterized by Gupta et al. (2006)
Expression of intermediate mesodermal markers (OSR1, WT1, PAX2, SIX2), while not increased with RA4 or RA7 in suspension culture, was rapidly and strongly upregulated in monolayer culture on gelatin-coated dishes (). These findings are supported by immunocytochemical analyses () that showed increased PAX2 and decreased Vimentin expression in RA7 cultures when compared with controls over time; and mirror trends in PAX2 and Vimentin expression observed in developing monkey kidneys (). A similar trend was noted in monolayer culture on a laminin substrate (), although the response was delayed and less in magnitude. Similarly, expression of kidney progenitor markers (EYA1, LIM1, CD24) was increased 2- to 4-fold in monolayer culture on gelatin-coated dishes (). Of this set of markers, only CD24 was increased when the culture substrate was laminin (). Since LIM1 was shown to be important in formation of the cranium (Shawlot and Behringer, 1995
), and EYA1 (Grifone et al., 2004
) and CD24 (Pirruccello and LeBien, 1986
) are expressed in muscle and lymphatic tissue, respectively, the data presented here must be interpreted with caution in terms of differentiation toward definitive renal phenotypes. In order to ensure renal cells for regenerative medicine purposes a combination of markers representative of specific stages of kidney differentiation are necessary to ensure lineage specificity. Methods for selecting these different populations will be important for exploring new regenerative techniques in animal models.
Extracellular matrix (ECM) molecules are important in kidney development and influence the physical organization of the cells, modulate signal transduction pathways, or regulate cell growth and proliferation through growth factor interactions (Kanwar et al., 2004
). Laminin, an adhesive glycoprotein, has been shown to play a role in epithelial and endothelial migration, proliferation, and function while collagen family proteins act as structural and regulatory proteins in a wide variety of mammalian tissues. In knockout mouse experiments, laminin and collagen have been shown to regulate formation and function of the glomerulus, as well as other related structures, and play crucial roles in the formation of the glomerular basement membrane (Lelongt and Ronco, 2003
). In the present studies, hESC differentiated in monolayer culture on substrates of gelatin (a heterogenous mixture composed primarily of collagen) or laminin were more responsive than hESC differentiated in suspension cultures as EBs. These findings support the idea that signaling through ECM molecules is important in directing early kidney differentiation.
RA, the active form of vitamin A, has a critical role in vertebrate embryonic development and has been shown to have wide-ranging effects on many anatomical areas such as the heart, brain, sensory systems, developing limbs, and respiratory system (Ross et al., 2001
; Zile, 2001
). The morphogenic properties of RA are likely mediated through tissue concentration gradients achieved in concert with distributions of various receptor isotypes and synthetic and catabolic enzymes in the developing embryo, and are perhaps best characterized in the emerging retina (Ross et al., 2001
). In the embryonic kidney, RA has been shown to regulate c-ret expression and ureteric bud branching morphogenesis (Mendelsohn et al., 1994
; Moreau et al., 1998
) in the mouse and rat.
Other cytokines/growth factors considered important in the posterior patterning and survival of the intermediate mesoderm include the BMP family of growth factors (Bush et al., 2004
; Xiao et al., 2007
). BMP-7-deficient mice begin early nephrogenic inductive events but show a reduction in the extent of branching morphogenesis, mesenchymal condensation, epithelial formation, and glomerular development (Dudley et al., 1995
; Luo et al., 1995
). Reduction of BMP-4 expression by its antagonist, gremlin, has been shown to be necessary for normal ureteric bud outgrowth and branching morphogenesis and for the establishment of the GDNF-RET signaling loop between the metanephric mesenchyme and the ureteric bud epithelium (Michos et al., 2007
). That BMP-4 is able to rescue BMP-7 mutant metanephric kidney development in mice suggests that, despite structural differences, BMP family members have redundant functions in kidney development (Oxburgh et al., 2005
). Notably, gene expression was similar for most genes when BMP-4 was substituted for BMP-7 in the present experiments providing additional in vitro
support for this hypothesis. Differential expression of CD24, OSR1, WT1, and LIM1 suggests that BMP-7 may promote more specific effects on this subset of genes.
In order to consider hESC for transplant purposes, graft purity will be necessary to ensure that contamination with undifferentiated cells does not occur and that only those cells directed towards a renal lineage are used for transplantation (Eiges et al., 2001
; Halme and Kessler, 2006
). Studies that focused on in vitro
differentiation include those in mice where an enriched population of mouse ESC was obtained by sorting for brachyury. Selected cells were noted to integrate into the nephrogenic zone in metanephric kidney organ culture and in the proximal tubules in newborn in vivo
transplant experiments (Vigneau et al., 2007
). Mouse EBs have been shown to express markers of the mature kidney such as podocin, nephrin, and Wt1 in later stages of cell culture (Kramer et al., 2006
). The addition of hepatocyte growth factor and Activin-A to mouse ESC transfected with Wnt4 cDNA was also shown to increase expression of Wt1, Wnt4, and Aqp2
in the later stages of mouse EB differentiation (Kobayashi et al., 2005
). Further, these cells were found to contribute to Aqp2
-positive tubules after injection into the renal cortex of host mice (Kobayashi et al., 2005
). Likewise, as noted above, the addition of RA, Activin-A, and BMP-7 to mouse ESC culture medium resulted in increased expression of early renal markers including Pax2 and Wt1, and enhanced efficiency of incorporation into tubule epithelia following injection into embryonic kidney cultures (Kim and Dressler, 2005
In summary, these studies have focused on spontaneous and directed differentiation of hESC toward renal lineages using a combination of factors. Taken together, these data support the interpretation that monolayer RA7 culture conditions may contribute to increased, but not definitive, differentiation of hESC along the renal pathway. Future investigations of renal ontogeny in monkeys with other developmental markers may illuminate critical branch points of renal developmental pathways and guide enhancement of renal differentiation protocols for hESC. Conversely, further investigations utilizing hESC as a model for mammalian kidney development will enhance our understanding of kidney precursor specification, and will be necessary for the development of new cell-based therapies for the treatment of human disease.