Abstract:Background
In diabetic kidney disease, high glucose damages specialized cells called podocytes that filter blood in the glomerulus. In vitro culture of podocytes is crucial for modeling of diabetic nephropathy and genetic podocytopathies and to complement animal studies. Recently, several methods have been published to derive podocytes from human-induced pluripotent stem cells (iPSCs) by directed differentiation. However, these methods have major variations in media composition and have not bee… Show more
“…Therefore, the same experiment was performed with two other antibodies, which were also found to be expressed in the nucleus (Supplementary Fig. S7 ), and the results were consistent with previous studies 26 , 37 . Figure 2 Identification of podocytes within glomerular cells cultured in vitro.…”
Podocytes are specialized terminally differentiated cells in the glomerulus that are the primary target cells in many glomerular diseases. However, the current podocyte cell lines suffer from prolonged in vitro differentiation and limited survival time, which impede research progress. Therefore, it is necessary to establish a cell line that exhibits superior performance and characteristics. We propose a simple protocol to obtain an immortalized mouse podocyte cell (MPC) line from suckling mouse kidneys. Primary podocytes were cultured in vitro and infected with the SV40 tsA58 gene to obtain immortalized MPCs. The podocytes were characterized using Western blotting and quantitative real-time PCR. Podocyte injury was examined using the Cell Counting Kit-8 assay and flow cytometry. First, we successfully isolated an MPC line and identified 39 °C as the optimal differentiation temperature. Compared to undifferentiated MPCs, the expression of WT1 and synaptopodin was upregulated in differentiated MPCs. Second, the MPCs ceased proliferating at a nonpermissive temperature after day 4, and podocyte-specific proteins were expressed normally after at least 15 passages. Finally, podocyte injury models were induced to simulate podocyte injury in vitro. In summary, we provide a simple and popularized protocol to establish a conditionally immortalized MPC, which is a powerful tool for the study of podocytes.
“…Therefore, the same experiment was performed with two other antibodies, which were also found to be expressed in the nucleus (Supplementary Fig. S7 ), and the results were consistent with previous studies 26 , 37 . Figure 2 Identification of podocytes within glomerular cells cultured in vitro.…”
Podocytes are specialized terminally differentiated cells in the glomerulus that are the primary target cells in many glomerular diseases. However, the current podocyte cell lines suffer from prolonged in vitro differentiation and limited survival time, which impede research progress. Therefore, it is necessary to establish a cell line that exhibits superior performance and characteristics. We propose a simple protocol to obtain an immortalized mouse podocyte cell (MPC) line from suckling mouse kidneys. Primary podocytes were cultured in vitro and infected with the SV40 tsA58 gene to obtain immortalized MPCs. The podocytes were characterized using Western blotting and quantitative real-time PCR. Podocyte injury was examined using the Cell Counting Kit-8 assay and flow cytometry. First, we successfully isolated an MPC line and identified 39 °C as the optimal differentiation temperature. Compared to undifferentiated MPCs, the expression of WT1 and synaptopodin was upregulated in differentiated MPCs. Second, the MPCs ceased proliferating at a nonpermissive temperature after day 4, and podocyte-specific proteins were expressed normally after at least 15 passages. Finally, podocyte injury models were induced to simulate podocyte injury in vitro. In summary, we provide a simple and popularized protocol to establish a conditionally immortalized MPC, which is a powerful tool for the study of podocytes.
“…Importantly, our method reduces the number and concentrations of morphogens compared to other protocols, while maintaining high yields of IM cells. Many established protocols require various morphogens or concentrations ( Yucer et al, 2017 ; Miyazaki et al, 2018 ; Knarston et al, 2020 ; Bejoy et al, 2022 ; Gong et al, 2022 ), leading to inconsistent target cell generation and lacking a lineage-directed approach. By replicating features of embryogenesis not captured in previous protocols, our method offers a more robust, reproducible, and efficient approach for organoid development and disease modeling.…”
Section: Discussionmentioning
confidence: 99%
“…However, human pluripotent stem cells (hPSC) offer powerful tool to overcome these limitations. Recent studies have demonstrated the generation of various cell types from hPSC, including IM-derived cells from hPSC ( Yucer et al, 2021 ; 2017 ; Miyazaki et al, 2018 ; Knarston et al, 2020 ; Bejoy et al, 2022 ; Gong et al, 2022 ). This ability of hPSC to recapitulate different stages of human embryonic development, mimicking the natural developmental sequence, has revolutionized our understanding of human biology and disease.…”
Section: Introductionmentioning
confidence: 99%
“…Several methods have been developed to derive mature tissues from the IM lineage using hPSC. These protocols have successfully generated tissue, such as fallopian tube epithelium ( Yucer et al, 2021 ; 2017 ), endometrial stromal fibroblasts ( Miyazaki et al, 2018 ), podocytes ( Bejoy et al, 2022 ), Müllerian duct-like cells ( Gong et al, 2022 ) or gonad and testis cell ( Knarston et al, 2020 ; Yucer et al, 2017 showed the generation of TBXT+ (T-box transcription factor T, protein) (BRACHYURY)/MIXL1+ (Mix paired-like homeobox, protein) PPS and OSR1+ (Odd-skipped related transcription factor 1, protein)/GATA3+ (GATA binding protein 3, protein)/PAX2+ (paired box 2, protein) IM cells. In their study, human induced pluripotent stem cells (hiPSC) were initially exposed to 100 ng/mL human recombinant Activin A and 3 μM CHIR99021 for 48 h to differentiate into mesoderm, followed by IM induction using 100 ng/mL BMP4 and 3 μM CHIR99021 for 48 h. Knarston et al, 2020 described PPS differentiation with 3 μM CHIR99021 for 96 h and IM lineage OSR1+/LHX1+ (LIM homeobox 1, protein)/PAX2+ with 10 ng/mL BMP4, 1 μg/mL Heparin and 200 ng/mL FGF9 for 72 h. Bejoy et al, 2022 have reported an accelerated method by differentiating hPSC into MIXL1+ primitive streak cells with 100 ng/mL human Activin A and 3 μM CHIR99021 for 48 h, and IM PAX8+(paired box 8, protein) cells with 8 μM CHIR99021 for 72 h. Gong et al, 2022 treated hPSC with 5 μM CHIR99021 for 36 h, identifying successful induction of TBXT+/MIXL1+ mesendodermal cells; then 100 ng/mL basic fibroblast growth factor (bFGF) and 10 nM Retinoic acid (RA) for OSR1+/PAX2+/LHX1+IM cell differentiation for 72 h.…”
Several differentiation protocols have enabled the generation of intermediate mesoderm (IM)-derived cells from human pluripotent stem cells (hPSC). However, the substantial variability between existing protocols for generating IM cells compromises their efficiency, reproducibility, and overall success, potentially hindering the utility of urogenital system organoids. Here, we examined the role of high levels of Nodal signaling and BMP activity, as well as WNT signaling in the specification of IM cells derived from a UCSD167i-99-1 human induced pluripotent stem cells (hiPSC) line. We demonstrate that precise modulation of WNT and BMP signaling significantly enhances IM differentiation efficiency. Treatment of hPSC with 3 μM CHIR99021 induced TBXT+/MIXL1+ mesoderm progenitor (MP) cells after 48 h of differentiation. Further treatment with a combination of 3 μM CHIR99021 and 4 ng/mL BMP4 resulted in the generation of OSR1+/GATA3+/PAX2+ IM cells within a subsequent 48 h period. Molecular characterization of differentiated cells was confirmed through immunofluorescence staining and RT-qPCR. Hence, this study establishes a consistent and reproducible protocol for differentiating hiPSC into IM cells that faithfully recapitulates the molecular signatures of IM development. This protocol holds promise for improving the success of protocols designed to generate urogenital system organoids in vitro, with potential applications in regenerative medicine, drug discovery, and disease modeling.
“…iPSCs are pluripotent stem cells reprogrammed from terminally differentiated somatic cells, such as fibroblasts, keratinocytes, extraembryonic tissues, umbilical cord blood and peripheral blood cells. With a strong differentiation capacity, iPSCs could be induced to differentiate into renal tubular cells and podocytes ( 232 ). ESCs are multipotent cells derived from the cell mass within the blastocyst which can be induced to the cells of the kidney lineage.…”
Diabetic kidney disease (DKD) is a chronic complication of diabetes and the leading cause of end-stage renal disease (ESRD) worldwide. Currently, there are limited therapeutic drugs available for DKD. While previous research has primarily focused on glomerular injury, recent studies have increasingly emphasized the role of renal tubular injury in the pathogenesis of DKD. Various factors, including hyperglycemia, lipid accumulation, oxidative stress, hypoxia, RAAS, ER stress, inflammation, EMT and programmed cell death, have been shown to induce renal tubular injury and contribute to the progression of DKD. Additionally, traditional hypoglycemic drugs, anti-inflammation therapies, anti-senescence therapies, mineralocorticoid receptor antagonists, and stem cell therapies have demonstrated their potential to alleviate renal tubular injury in DKD. This review will provide insights into the latest research on the mechanisms and treatments of renal tubular injury in DKD.
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