Renal ontogeny in the rhesus monkey (Macaca mulatta) and directed differentiation of human embryonic stem cells towards kidney precursors

Abstract

The development of the metanephric kidney was studied immunohistochemically across gestation in monkeys to identify markers of cell specification, and to aid in developing experimental paradigms for renal precursor differentiation from human embryonic stem cells (hESC). PAX2, an important kidney developmental marker, was expressed at the tips of the ureteric bud, in the surrounding condensing mesenchyme, and in the renal vesicle. Vimentin, a mesenchymal and renal marker, was strongly expressed in the metanephric blastema then found to be limited to the glomerulus and interstitial cells of the medulla and cortex. A model of gene expression based on human and nonhuman primate renal ontogeny was developed and incorporated into studies of hESC differentiation. Spontaneous hESC differentiation revealed markers of metanephric mesenchyme (OSR1, PAX2, SIX2, WT1) that increased over time, followed by upregulation of kidney precursor markers (EYA1, LIM1, CD24). Directed hESC differentiation was also evaluated with the addition of Retinoic Acid, Activin-A, and BMP-4 or BMP-7, and using different culture substrate conditions. Of the culture substrates studied, gelatin most closely recapitulated the anticipated directed developmental pattern of renal gene expression. No differences were found when BMP-4 and BMP-7 were compared to baseline conditions. PAX2 and Vimentin immunoreactivity in differentiating hESC was also similar to the renal precursor patterns reported for human fetal kidneys and findings described in rhesus monkeys. The results of these studies: (1) provide additional data to support that rhesus monkey kidney development parallels that of humans, and (2) provide a useful model for hESC directed differentiation towards renal precursors.

Rapid and Efficient Differentiation of Human Pluripotent Stem Cells into Intermediate Mesoderm That Forms Tubules Expressing Kidney Proximal Tubular Markers

“…Regardless of the duration of CHIR pretreatment, the proportion of cells expressing PAX2 significantly decreased after day 4 of differentiation, with ,10% of cells at day 7 retaining PAX2 expression. Because previous studies have identified a role for BMP7 in inducing IM cells, 5,14,16,17 we next determined whether the addition of BMP7 to FGF2 and RA could enhance IM cell differentiation. In contrast to these other reports, we found that the addition of BMP7 significantly To determine the reproducibility of our protocol in different hPSC lines, we tested the combination of CHIR induction for 36 hours followed by the addition of FGF2 and RA in three hESC lines and two hiPSC lines.…”

“…Although the differentiation of hPSCs into cells of the cardiac, hepatic, pancreatic, and neuronal lineages has been widely reported, relatively few previous studies have attempted to derive cells of the kidney lineage from hPSCs. [16][17][18][19] An alternative to directed differentiation was recently demonstrated by means of direct reprogramming of immortalized human kidney cells into nephron progenitor-like cells; however, the efficiency of integration into kidney explant cultures was reportedly low. 63 Mae and colleagues demonstrated induction of an intermediate mesoderm population using a stepwise combination of CHIR, activin, and BMP7 signaling in engineered OSR1-GFP hiPSC cell lines, achieving efficiencies of .90% of OSR1-GFP + cells after 11-18 days of differentiation.…”

“…[16][17][18][19] These previous reports have produced cells that share characteristics expected of human kidney progenitor or epithelial cells, although the identities of these differentiated cells have yet to be conclusively verified. In addition, the efficiencies of these protocols for generating cells of the renal lineage are low, necessitating the use of cell sorting to enrich populations of cells using markers that are not entirely specific to the kidney.…”

Renal Tissue Engineering with Decellularized Rhesus Monkey Kidneys: Age-Related Differences

“…13,17 Slides were washed in PBS before heat-mediated antigen retrieval in citrate buffer (pH 6; Invitrogen) was performed. After cooling, decreasing concentrations of warm citrate buffer in PBS was applied followed by incubation with Background Sniper (BioCare Medical), which was added to each slide for 15 min.…”

The use of bioreactors as in vitro models in pharmaceutical research

“…Studies into human ESCs differentiating into renal cells have not been widely undertaken, however preliminary studies have shown integration of undifferentiated and mesoderm-derived ESCs into the stroma and developing nephrons [82]. They have also been shown to express a wide range of early renal markers, and exhibit up-regulation of kidney precursor markers such as EYA1, LIM1, CD24 [83]. The generation of the different types of renal cells from human ESCs however still needs to be thoroughly investigated [82].…”