Abstract:Generating human podocytes in vitro could offer a unique opportunity to study human diseases. Here, we describe a simple and efficient protocol for obtaining functional podocytes in vitro from human induced pluripotent stem cells. Cells were exposed to a three-step protocol, which induced their differentiation into intermediate mesoderm, then into nephron progenitors and, finally, into mature podocytes. After differentiation, cells expressed the main podocyte markers, such as synaptopodin, WT1, α-Actinin-4, P-… Show more
“…In an effort to generate tools for both drug discovery and drug safety within the area of kidney disease, we aimed to develop an in vitro model for glomerular health using hiPSCs. To date, some recent progress has been made toward this end, [1][2][3][4][5]28 but there are still no established functional tools mimicking the human situation in the screening format required for drug discovery.…”
Section: Discussionmentioning
confidence: 99%
“…The induced pluripotent stem cell (iPSC) technology has made it possible to access and modify cells of a wide variety of origins that previously have been difficult or nearly impossible to study. Within the field of nephrology, some recent advances have been made in creating 3-dimensional (3D) organoid cultures, [1][2][3][4][5] but there are still limited functional tools for drug discovery available.…”
Development of physiologically relevant cellular models with strong translatability to human pathophysiology is critical for identification and validation of novel therapeutic targets. Herein we describe a detailed protocol for generation of an advanced 3-dimensional kidney cellular model using induced pluripotent stem cells, where differentiation and maturation of kidney progenitors and podocytes can be monitored in live cells due to CRISPR/Cas9-mediated fluorescent tagging of kidney lineage markers (SIX2 and NPHS1). Utilizing these cell lines, we have refined the previously published procedures to generate a new, higher throughput protocol suitable for drug discovery. Using paraffin-embedded sectioning and whole-mount immunostaining, we demonstrated that organoids grown in suspension culture express key markers of kidney biology (WT1, ECAD, LTL, nephrin) and vasculature (CD31) within renal cortical structures with microvilli, tight junctions and podocyte foot processes visualized by electron microscopy. Additionally, the organoids resemble the adult kidney transcriptomics profile, thereby strengthening the translatability of our in vitro model. Thus, development of human nephron-like structures in vitro fills a major gap in our ability to assess the effect of potential treatment on key kidney structures, opening up a wide range of possibilities to improve clinical translation.
“…In an effort to generate tools for both drug discovery and drug safety within the area of kidney disease, we aimed to develop an in vitro model for glomerular health using hiPSCs. To date, some recent progress has been made toward this end, [1][2][3][4][5]28 but there are still no established functional tools mimicking the human situation in the screening format required for drug discovery.…”
Section: Discussionmentioning
confidence: 99%
“…The induced pluripotent stem cell (iPSC) technology has made it possible to access and modify cells of a wide variety of origins that previously have been difficult or nearly impossible to study. Within the field of nephrology, some recent advances have been made in creating 3-dimensional (3D) organoid cultures, [1][2][3][4][5] but there are still limited functional tools for drug discovery available.…”
Development of physiologically relevant cellular models with strong translatability to human pathophysiology is critical for identification and validation of novel therapeutic targets. Herein we describe a detailed protocol for generation of an advanced 3-dimensional kidney cellular model using induced pluripotent stem cells, where differentiation and maturation of kidney progenitors and podocytes can be monitored in live cells due to CRISPR/Cas9-mediated fluorescent tagging of kidney lineage markers (SIX2 and NPHS1). Utilizing these cell lines, we have refined the previously published procedures to generate a new, higher throughput protocol suitable for drug discovery. Using paraffin-embedded sectioning and whole-mount immunostaining, we demonstrated that organoids grown in suspension culture express key markers of kidney biology (WT1, ECAD, LTL, nephrin) and vasculature (CD31) within renal cortical structures with microvilli, tight junctions and podocyte foot processes visualized by electron microscopy. Additionally, the organoids resemble the adult kidney transcriptomics profile, thereby strengthening the translatability of our in vitro model. Thus, development of human nephron-like structures in vitro fills a major gap in our ability to assess the effect of potential treatment on key kidney structures, opening up a wide range of possibilities to improve clinical translation.
“…Urinary renal progenitor cells have the potential to be used as a non-invasive source for studying personalized genetic kidney disease, but the number of progenitor cells present in urine varies and hampers the success rate of urine-derived progenitor and associated podocyte cultures. To overcome this hurdle, the use of (patient-derived) induced pluripotent stem cells (iPSC) and differentiation towards podocytes is a promising approach [21][22][23][24]. Tanigawa and co-workers successfully reprogrammed skin fibroblasts isolated from a newborn diagnosed with congenital NS into iPSC [12].…”
Section: Functional Assessment Of Genetic Variantsmentioning
confidence: 99%
“…2). Using either 2D or 3D culture approaches, and varying the choice and/or timing of growth factors, result in the induction of one renal cell type (e.g., podocyte-like cells) [15,21,55,56] or in self-organizing kidney organoids consisting of segmented nephrons [22,23,[57][58][59]. These protocols are extensively reviewed by Soo et al [60].…”
Nephrotic syndrome is a heterogeneous disease, and one of the most frequent glomerular disorders among children. Depending on the etiology, it may result in end-stage renal disease and the need for renal replacement therapy. A dysfunctional glomerular filtration barrier, comprising of endothelial cells, the glomerular basement membrane and podocytes, characterizes nephrotic syndrome. Podocytes are often the primary target cells in the pathogenesis, in which not only the podocyte function but also their crosstalk with other glomerular cell types can be disturbed due to a myriad of factors. The pathophysiology of nephrotic syndrome is highly complex and studying molecular mechanisms in vitro requires state-of-the-art cell-based models resembling the in vivo situation and preferably a fully functional glomerular filtration barrier. Current advances in stem cell biology and microfluidic platforms have heralded a new era of three-dimensional (3D) cultures that might have the potential to recapitulate the glomerular filtration barrier in vitro. Here, we highlight the molecular basis of nephrotic syndrome and discuss requirements to accurately study nephrotic syndrome in vitro, including an overview of specific podocyte markers, cutting-edge stem cell organoids, and the implementation of microfluidic platforms. The development of (patho) physiologically relevant glomerular models will accelerate the identification of molecular targets involved in nephrotic syndrome and may be the harbinger of a new era of therapeutic avenues.
“…Currently, there are several studies that have reported the differentiation of hPSCs into cells of the kidney lineage [3–7, 34, 35, 64]. Kidneys consist of many different cell types in the nephron, vasculature and interstitial compartments.…”
Section: Comparison Of Kidney Organoid Differentiation Protocolsmentioning
Human pluripotent stem cells (hPSCs) are attractive sources for regenerative medicine and disease modeling in vitro. Directed hPSC differentiation approaches have derived from knowledge of cell development in vivo rather than from stochastic cell differentiation. Moreover, there has been great success in the generation of 3-dimensional organ-buds termed “organoids” from hPSCs; these consist of a variety of cell types in vitro to mimic organs in vivo. The organoid bears great potential in the study of human diseases in vitro especially when combined with CRISPR/Cas9-based genome editing approaches. We summarize the current literature describing organoid studies with a special focus on kidney organoids, and discuss goals and future opportunities for organoid-based studies.
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