In this study, we demonstrate that it is possible to generate renal cells from human ES cells in vitro. Initially, we established that the markers WT1, cadherin 16, and podocalyxin recognize structures derived from the MM and UB in the developing human embryo. While the pluripotent nature of human ES cells is well accepted, to date there have been 2 reports of the formation of kidney-like structures from human ES cell-derived teratomas [23
]. Only one of these reports presented immunohistochemistry for markers of renal structures (WT1 and NCAM) [25
]. We have confirmed and extended these findings using the renal markers WT1, cadherin 16, and podocalyxin and show that human ES cells indeed have the ability to differentiate into structures resembling fetal glomeruli. In addition, we also extend these results to validate that the structures observed in the teratomas are of the renal lineage.
In order to achieve the long-term goal of utilizing human ES cells in cell replacement therapy for renal disease, methods for obtaining renal cells from human ES cells in a controlled and reproducible manner are required. To date, there has been only one study involving the directed differentiation of human ES cells toward the renal lineage, which indicated that it is possible to detect an up-regulation of MM-related transcripts (OSR1, PAX2, SIX2, and WT1) and genes associated with kidney precursors (EYA1, LIM1,
) in human ES cell colonies cultured in the presence of retinoic acid, activin A, and BMP4 or BMP7 on laminin or gelatin substrates [27
]. In the present study, we employed a strategy of serum concentration reduction and feeder depletion to induce differentiation of human ES cells. As noted, serum reduction in combination with growth factors such as activin A [35
] or in a co-culture system [34
] has previously been used to differentiate human ES cells toward the cardiomyocyte and pancreatic lineages, respectively. In the case of endoderm differentiation, this effect at least partially relies on the reduction of insulin-like growth factor (IGF), usually present in the serum, which is thought to antagonize differentiation of human ES cells via the activation of the phosphotidylinositol-3-kinase (PI3K) signaling pathway [45
]. While the effect of this pathway on differentiation toward other lineages has not been investigated, we speculate that similar factors are present in serum and either have an inhibitory effect on differentiation, a stimulatory effect on the maintenance of pluripotency, or both. As with most protocols involving human ES cell differentiation, the resulting cell population represented a heterogeneous mixture of cell types. The markers CD24, podocalyxin, and GCTM2 were selected for use in FACS to isolate renal cells from this mixture of cells. Individually, neither CD24 nor podocalyxin are restricted in their expression to the developing kidney. However, using them in combination with GCTM2 exclusion enables a subset of differentiated cells containing the renal precursor cells of interest to be isolated from a global heterogeneous population of differentiated and pluripotent cells. We therefore isolated 3 fractions of cells CD24+
(++Low), and CD24+
/GCTM2-(++−). While each of these fractions are still heterogeneous, we hypothesized that the ++Hi fraction would contain the highest proportion of pluripotent cells, the ++Low fraction contains cells that have begun to commit to a mesoderm fate, while the ++− fraction contains the renal cells of interest. We confirmed this hypothesis using quantitative gene expression analysis and showed that the ++Low and ++− fractions have indeed proceeded along the pathway of differentiation based on their down-regulation of the stemness genes, OCT4
With particular regards to HES-4, the observed up-regulation of LHX1 transcripts in the ++Low but not the ++− fraction lends further weight to the hypothesis that this fraction contains less differentiated cells than ++− since Lim1 is critical for intermediate mesoderm formation in the mouse. While the quantitative PCR readout indicated that HES-4 is the cell line that is most amenable to the differentiation regime, similar trends in gene expression were observed in the other 2 human ES cell lines (Supplementary Table 1
).This variation in responsiveness of different cell lines is not uncommon and has been reported with differentiation protocols aimed at obtaining pancreatic endocrine cells from human ES cells [46
]. Notably, these gene expression results are in line with reports by D'Amour and colleagues (2005) who showed that culture of human ES cells for 4 days in the presence of low serum results in high expression of mesoderm genes.
As organogenesis is a complex process that involves the expression of a multitude of genes, evidence of lineage commitment requires larger-scale characterization via transcriptome profiling. Our microarrays provided strong evidence of a MM-like population within the FACS-isolated fractions of differentiated human ES cells. From our gene ontology results, the observed up-regulation of kidney-associated genes and the significant decrease in transcripts associated with neural and muscular development in the ++− fraction relative to spontaneously differentiated human ES cells is particularly encouraging, as the induction of neural differentiation is a default pathway that occurs autonomously when inhibitory signals are removed [47
]. With respect to muscular lineages, while it appears that the sorting strategy may also be selecting against cells that have differentiated along muscular lineages, the ontology analysis for markers of muscle development includes genes associated with skeletal, cardiac, visceral, and smooth muscles based on cluster and gene ontology analyses. The efficacy of our sorting strategy is also promising based on results of our cluster analysis, which show that the ++− fraction contains cells that have an increased expression of genes associated with specific compartments of the kidney during development.
Overall, our results suggest the selection of a population enriched in cells adopting a phenotype derived from intermediate mesoderm, the source tissue for the kidney. In addition, this fraction showed differential expression of markers previously associated with the MM. As noted previously, this tissue gives rise to both the nephrons and components of the renal interstitium. The former target is the most critical for any potential renal regenerative option. There are many known transcription factors that differentiate between the MM fated to become nephron versus interstitium and while this differentiation and enrichment strategy showed up-regulation of MM markers, this did not include those specifically marking the nephron progenitors. For the MM to undergo a mesenchymal to epithelial transition to form a nephron, the cells must co-express WT1 and PAX2. While expression of each of these markers individually have been reported in a variety of nonrenal cell types, their co-expression during embryonic development is a strong indicator of nephron progenitor activity [22
]. We identified a small population of WT1+
cells within the ++− fraction suggesting that such progenitors were being induced at a low rate. What will be critical now is to optimize further selective strategies for the enrichment or selective culture of that nephron progenitor subfraction. Such a population would be invaluable as a source for the terminal differentiation of mature renal cells from human ES cells for toxicology screening and potentially for renal therapy.