Renal cells are used in basic research, disease models, tissue engineering, drug screening, and in vitro toxicology. The kidneys have highly differentiated and complicated structures, and have pivotal roles in many physiological processes, such as body fluid osmolality, regulation of fluid and electrolyte balance, regulation of acid-base balance, excretion of metabolic waste products and foreign chemicals, and production of hormones controlling blood pressure and erythropoiesis. Once damaged, kidneys rarely recover their functions. Renal cells (e.g. Podocytes and tubular cells) can regenerate to some extent following acute necrosis. However, kidneys generally do not regenerate in patients with chronic kidney diseases (Humphreys and Bonventre, 2007), leading to end-stage renal insufficiency. Chronic kidney disease (CKD) is a major cause of morbidity and mortality affecting 11% of the adult population in Western countries. People with CKD suffer from a substantial loss of quality of life. The pharmacoeconomic burden caused by this disease is very high, as there is a permanent shortage of donor kidneys for transplantation.
The mammalian kidney is derived from the intermediate mesoderm (IM), which gives rise to the nephric duct (ND), and the metanephric mesenchyme (MM). The ND gives rise to the collecting duct system, which is composed of two key cell types, principal cells, and intercalated cells. The MM specifies the cap mesenchyme (CM) and also gives rise to the stroma. The CM is the nephron progenitor population and differentiates in the renal vesicle via a mesenchyme-to-epithelial transition.
The nephron consists of a glomerular tuft or glomerulus, and a renal tubule. The glomerulus is a highly specialized filtration unit that separates waste products for excretion as urine. The filtration barrier between blood and urine in the glomerulus is provided by highly specialized, terminally differentiated cells termed podocytes.
Converging evidence suggests that damage to the podocytes is one of the key events triggering loss of renal function. Podocyte damage occurs secondary to hyperinsulinemia, hemodynamic mechanisms and other mechanisms. The progressive loss of podocytes leads to broad sclerosis of the glomeruli accompanied by increased proteinuria and reduction in the clearance function (Wiggins, 2007).
However, the underlying mechanisms of insulin resistance and loss of regenerative properties leading to pathophysiological changes in the nephron of the kidney are not completely understood. Thus there is a need for in vitro cell models to study the biology of renal diseases like CKD and to facilitate the development of new treatments.
Embryonic stem (ES) cells and patient specific induced pluripotent stem cells (iPSCs) are a potential source for the production of renal precursor cells and podocytes in large scale for regenerative medicine and disease modeling for drug discovery. With the induced pluripotent stem cells (iPSCs) technology (Takahashi, K. & Yamanaka, S., “Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors”, Cell 126, 663-676 (2006)) somatic cells can be reprogrammed to iPSCs by transduction of four defined factors (Sox2, Oct4, Klf4, c-20 Myc). The iPSC technology enables the generation of patient specific iPSCs, which can be differentiated into patient specific renal cells. These patient specific renal cells are useful for example in vitro modeling of the pathophysiology of renal disease such as Chronic Kidney Disease (CKD), Focal segmental glomerulosclerosis (FSGS), Membranoproliferative glomerulonephritis, Polycystic kidney disease (PKD) and diabetic nephropathy associated with Diabetes Type-2, or for the assessment of drug toxicity. One important prerequisite to attempt such in vitro disease modeling is the implementation of an efficient, robust and scalable differentiation system (Tiscornia et al., 2011).
Previous efforts to differentiate human PSCs into renal cells have not achieved scales and efficacies relevant for drug discovery campaigns or regenerative cell therapies, neither in humans (Batchelder et al., 2009; Lin et al., 2010; Mae et al., 2013; Narayanan et al., 2013; Song et al., 2012) or mice (Kim and Dressler, 2005; Mae et al., 2010; Morizane et al., 2009; Nishikawa et al., 2012; Ren et al., 2010). In addition, a major concern is the mal-differentiation of the cells into unwanted tissues or even the formation of teratomas. To avoid this danger, one must direct the cells to a state of differentiation that will on the one hand provide them with the potential to regenerate mature kidney cells of interest and on the other hand prevent mal-differentiation. This can be achieved by the controlled activation of the correct network of nephric transcription factors. Unfortunately, attaining this exact state of differentiation in vitro has proven to be quite difficult. Many attempts have been made to induce pluripotent cells in this manner, applying both growth factor combinations [bone morphogenetic protein (BMP)/Activin/Retinoic acid] and genetic approaches. However, most differentiation studies, even after successfully inducing renal lineage genes, failed to pinpoint the exact stage in nephrogenesis (IM, MM, CM) to which ESCs were differentiated along the renal lineage.
Therefore, a highly efficient and chemically defined method for stimulating the differentiation of human pluripotent stem cells into kidney lineages remains to be developed.
Mae et al. 2013 describe a protocol to differentiate human pluripotent stem cells into intermediate mesoderm cells which express Osr1 using defined induction steps in serum free media. The authors dissociated the undifferentiated cells with Accutase® to obtain a single layer of cells and induced the differentiation with Activin A, a GSK3 beta inhibitor and a ROCK kinase inhibitor in a first step and BMP7 and the GSK3 beta inhibitor in a second step. The authors obtained 90% Osr1 positive cells on day 11 only. Expression of PAX2, LIM1, WT1, CITED2, EYA1 and SALL1 (marker genes for the developing kidney, gonad and adrenal cortex) was observed after 18 days, indicating that the authors obtained a heterogeneous cell population of different cell types and cells in different differentiation stages.
Lin et al., 2010 describe the differentiation of human embryonic stem cells into mesodermal renal progenitor lineages by reducing serum concentration and feeder layer density for 14 days. The authors obtained a heterogeneous population of differentiated human embryonic cells which they fractionated by flow cytometry.
Batchelder et al., 2009 describe the direct differentiation of embryonic stem cells towards the renal lineage by culturing the embryonic stem cells with retinoic acid, activin A, BMP7 or BMP4 on laminin or gelatin substrates in a monolayer. They obtained cells with upregulated intermediate mesoderm marker genes (PAX2, SIX2, WT1 and OSR1) at day 4. However, markers for kidney precursors and markers of undifferentiated cells were also elevated at day 4. Batchelder et al do not show any further differentiation of this heterogeneous population into defined cell types. The differentiation of the embryonic stem cells is achieved through a stage with embryoid bodies, which generally limits reproducibility and standardization of the protocol.
Hence, prior art protocols for differentiation of human pluripotent stem cells into kidney percursors have major drawbacks: Firstly, most protocols result in a heterogenous population of cells and the absolute yield of defined renal precursor cells stably expressing metanephric mesenchyme markers is very low. In addition, the overall time needed to differentiate pluripotent stem cells into renal precursor cells by most known methods is very long. Many protocols require undefined elements such as medium conditioned with factors secreted by primary cells, co-cultures with feeder layers, which limit the standardization of these methods. In addition, many protocols rely on cell aggregates or embryoid bodies, which due to their heterogeneous nature constrain the reproducibility of these techniques.
Song et al. 2012 is the first reported protocol to differentiate human induced pluripotent stem cells into kidney podocytes. Following ten days of directed differentiation in medium supplemented with fetal bovine serum and different growth factors (BMP7, Activin-A and retinoic acid), the authors obtained iPS cells with a podocyte phenotype. They obtained cells expressing podocyte specific genes but also still expressing the metanephric mesenchymal genes PAX2 and WT1, indicating that the obtained podocytes are immature and not fully differentiated. Song et al do not describe any of the intermediate stages of the differentiation like the intermediate mesoderm or the metanephric mesenchyme.
None of the known protocols provide defined renal precursor cells that express Six2, WT1, and SALL1 with downregulation of the expression of PAX2, i.e. defined metanephric mesenchyme cells. None of the known protocols describe the differentiation of the renal precursor cell into podocytes.
In summary, there is no method that provides a defined population of renal precursor cells expressing metanephric mesenchyme markers at very high yield after only six days. In addition none of the known protocols provides fully functional and fully differentiated podocytes at very high yield after only 13 days.
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