Differentiation of human iPSCs into functional podocytes
Podocytes play a critical role in glomerular barrier function, both in health and disease. However, in vivo terminally differentiated podocytes are difficult to be maintained in in vitro culture. Induced pluripotent stem cells (iPSCs) offer the unique possibility for directed differentiation into ma...
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| Veröffentlicht in: | PloS one 2018-09, Vol.13 (9), p.e0203869 |
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| creator | Rauch, Caroline Feifel, Elisabeth Kern, Georg Murphy, Cormac Meier, Florian Parson, Walther Beilmann, Mario Jennings, Paul Gstraunthaler, Gerhard Wilmes, Anja |
| description | Podocytes play a critical role in glomerular barrier function, both in health and disease. However, in vivo terminally differentiated podocytes are difficult to be maintained in in vitro culture. Induced pluripotent stem cells (iPSCs) offer the unique possibility for directed differentiation into mature podocytes. The current differentiation protocol to generate iPSC-derived podocyte-like cells provides a robust and reproducible method to obtain podocyte-like cells after 10 days that can be employed in in vitro research and biomedical engineering. Previous published protocols were improved by testing varying differentiation media, growth factors, seeding densities, and time course conditions. Modifications were made to optimize and simplify the one-step differentiation procedure. In contrast to earlier protocols, adherent cells for differentiation were used, the use of fetal bovine serum (FBS) was reduced to a minimum, and thus ß-mercaptoethanol could be omitted. The plating densities of iPSC stocks as well as the seeding densities for differentiation cultures turned out to be a crucial parameter for differentiation results. Conditionally immortalized human podocytes served as reference controls. iPSC-derived podocyte-like cells showed a typical podocyte-specific morphology and distinct expression of podocyte markers synaptopodin, podocin, nephrin and WT-1 after 10 days of differentiation as assessed by immunofluorescence staining or Western blot analysis. qPCR results showed a downregulation of pluripotency markers Oct4 and Sox-2 and a 9-fold upregulation of the podocyte marker synaptopodin during the time course of differentiation. Cultured podocytes exhibited endocytotic uptake of albumin. In toxicological assays, matured podocytes clearly responded to doxorubicin (Adriamycin™) with morphological alterations and a reduction in cell viability after 48 h of incubation. |
| doi_str_mv | 10.1371/journal.pone.0203869 |
| format | Article |
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However, in vivo terminally differentiated podocytes are difficult to be maintained in in vitro culture. Induced pluripotent stem cells (iPSCs) offer the unique possibility for directed differentiation into mature podocytes. The current differentiation protocol to generate iPSC-derived podocyte-like cells provides a robust and reproducible method to obtain podocyte-like cells after 10 days that can be employed in in vitro research and biomedical engineering. Previous published protocols were improved by testing varying differentiation media, growth factors, seeding densities, and time course conditions. Modifications were made to optimize and simplify the one-step differentiation procedure. In contrast to earlier protocols, adherent cells for differentiation were used, the use of fetal bovine serum (FBS) was reduced to a minimum, and thus ß-mercaptoethanol could be omitted. The plating densities of iPSC stocks as well as the seeding densities for differentiation cultures turned out to be a crucial parameter for differentiation results. Conditionally immortalized human podocytes served as reference controls. iPSC-derived podocyte-like cells showed a typical podocyte-specific morphology and distinct expression of podocyte markers synaptopodin, podocin, nephrin and WT-1 after 10 days of differentiation as assessed by immunofluorescence staining or Western blot analysis. qPCR results showed a downregulation of pluripotency markers Oct4 and Sox-2 and a 9-fold upregulation of the podocyte marker synaptopodin during the time course of differentiation. Cultured podocytes exhibited endocytotic uptake of albumin. In toxicological assays, matured podocytes clearly responded to doxorubicin (Adriamycin™) with morphological alterations and a reduction in cell viability after 48 h of incubation.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0203869</identifier><identifier>PMID: 30222766</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Adherent cells ; Biology and Life Sciences ; Biomarkers ; Biomedical engineering ; Biomedical materials ; Cell culture ; Culture media ; Diabetes ; Differentiation ; Doxorubicin ; Fetal calf serum ; Growth factors ; Immunofluorescence ; In vivo methods and tests ; Incubation ; Inhibitory postsynaptic potentials ; Kidney diseases ; Kinases ; Legal medicine ; Morphology ; Oct-4 protein ; Physical Sciences ; Physiology ; Pluripotency ; Product safety ; Research and Analysis Methods ; Stem cells ; Toxicology</subject><ispartof>PloS one, 2018-09, Vol.13 (9), p.e0203869</ispartof><rights>2018 Rauch et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2018 Rauch et al 2018 Rauch et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c526t-1d5e8ff5b92ea66f653f7fce803a2b55bb6be88e59b07ce7b50ae6c062bf4fe33</citedby><cites>FETCH-LOGICAL-c526t-1d5e8ff5b92ea66f653f7fce803a2b55bb6be88e59b07ce7b50ae6c062bf4fe33</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6141081/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6141081/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,315,729,782,786,866,887,2104,2930,23873,27931,27932,53798,53800</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/30222766$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Dryer, Stuart E.</contributor><creatorcontrib>Rauch, Caroline</creatorcontrib><creatorcontrib>Feifel, Elisabeth</creatorcontrib><creatorcontrib>Kern, Georg</creatorcontrib><creatorcontrib>Murphy, Cormac</creatorcontrib><creatorcontrib>Meier, Florian</creatorcontrib><creatorcontrib>Parson, Walther</creatorcontrib><creatorcontrib>Beilmann, Mario</creatorcontrib><creatorcontrib>Jennings, Paul</creatorcontrib><creatorcontrib>Gstraunthaler, Gerhard</creatorcontrib><creatorcontrib>Wilmes, Anja</creatorcontrib><title>Differentiation of human iPSCs into functional podocytes</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Podocytes play a critical role in glomerular barrier function, both in health and disease. However, in vivo terminally differentiated podocytes are difficult to be maintained in in vitro culture. Induced pluripotent stem cells (iPSCs) offer the unique possibility for directed differentiation into mature podocytes. The current differentiation protocol to generate iPSC-derived podocyte-like cells provides a robust and reproducible method to obtain podocyte-like cells after 10 days that can be employed in in vitro research and biomedical engineering. Previous published protocols were improved by testing varying differentiation media, growth factors, seeding densities, and time course conditions. Modifications were made to optimize and simplify the one-step differentiation procedure. In contrast to earlier protocols, adherent cells for differentiation were used, the use of fetal bovine serum (FBS) was reduced to a minimum, and thus ß-mercaptoethanol could be omitted. 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critical role in glomerular barrier function, both in health and disease. However, in vivo terminally differentiated podocytes are difficult to be maintained in in vitro culture. Induced pluripotent stem cells (iPSCs) offer the unique possibility for directed differentiation into mature podocytes. The current differentiation protocol to generate iPSC-derived podocyte-like cells provides a robust and reproducible method to obtain podocyte-like cells after 10 days that can be employed in in vitro research and biomedical engineering. Previous published protocols were improved by testing varying differentiation media, growth factors, seeding densities, and time course conditions. Modifications were made to optimize and simplify the one-step differentiation procedure. In contrast to earlier protocols, adherent cells for differentiation were used, the use of fetal bovine serum (FBS) was reduced to a minimum, and thus ß-mercaptoethanol could be omitted. The plating densities of iPSC stocks as well as the seeding densities for differentiation cultures turned out to be a crucial parameter for differentiation results. Conditionally immortalized human podocytes served as reference controls. iPSC-derived podocyte-like cells showed a typical podocyte-specific morphology and distinct expression of podocyte markers synaptopodin, podocin, nephrin and WT-1 after 10 days of differentiation as assessed by immunofluorescence staining or Western blot analysis. qPCR results showed a downregulation of pluripotency markers Oct4 and Sox-2 and a 9-fold upregulation of the podocyte marker synaptopodin during the time course of differentiation. Cultured podocytes exhibited endocytotic uptake of albumin. In toxicological assays, matured podocytes clearly responded to doxorubicin (Adriamycin™) with morphological alterations and a reduction in cell viability after 48 h of incubation.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>30222766</pmid><doi>10.1371/journal.pone.0203869</doi><oa>free_for_read</oa></addata></record> |
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| subjects | Adherent cells Biology and Life Sciences Biomarkers Biomedical engineering Biomedical materials Cell culture Culture media Diabetes Differentiation Doxorubicin Fetal calf serum Growth factors Immunofluorescence In vivo methods and tests Incubation Inhibitory postsynaptic potentials Kidney diseases Kinases Legal medicine Morphology Oct-4 protein Physical Sciences Physiology Pluripotency Product safety Research and Analysis Methods Stem cells Toxicology |
| title | Differentiation of human iPSCs into functional podocytes |
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