This work was funded by the Interdisciplinary Center for Clinical Research (IZKF) of Friedrich-Alexander University Erlangen-N&#252;rnberg with the grant number M4-IZKF-F009 given to Janina M&#252;ller-Deile, and by Bundesministerium f&#252;r Bildung und Forschung (BMBF) under the project name STOP-FSGS-Speed Translation-Oriented Progress to Treat FSGS, grant number 01GM2202D given to Janina M&#252;ller-Deile. We thank Annalena Kraus for the support in taking SEM images.

Using Skin Biopsies to Generate Patient-Derived Podocytes

<ol>
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The authors have nothing to disclose.

This cell culture-based protocol combines the episomal reprogramming of human dermal fibroblasts into patient-specific hiPSCs and subsequent differentiation into hiPSC-derived podocytes. This allows us to study mutation-related alterations of podocytes from patients with genetic glomerular disease regarding podocyte injury. The protocol to reprogram dermal fibroblasts with an integration-free method by electroporation is adapted from the published work of Bang et al.32 and Okita et al.33. The protocol to differentiate podocytes from hiPSCs is adapted from the published protocol from Musah et al.28,34,35. There are already publications available describing the generation of podocytes from hiPSCs27,34,35. However, the protocol provided here is optimized and less expensive regarding the differentiation of hiPSCs into podocytes. Compared to the published protocol from Musah et al., we tested this protocol on different coating reagents, like vitronectin, laminin silk-511, and solubilized basement membrane matrix. The concentrations of vitronectin and laminin silk-511 could be decreased to 2.5 &#181;g/mL instead of 5 &#181;g/mL28,34,35. Furthermore, it was possible to decrease the concentrations of BMP7 and activin A-two very expensive growth factors-by 50%, from 100 ng/mL to 50 ng/mL.
This enables less expensive differentiation. Nephron progenitor cells from day 7 were still proliferating, and the possibility of freezing was shown before. We expanded these cells after thawing and before final differentiation in basic medium containing Dulbecco&#39;s modified Eagle medium (DMEM) and B27 for several days, decreasing the costs even further. In addition to the differentiation steps, this protocol describes the outgrowth of fibroblasts from skin biopsies with subsequent generation of patient-specific hiPSCs via episomal reprogramming. The combination of these two methods enables the generation of patient-specific podocytes. Therefore, a complete step-by-step protocol to generate patient-specific hiPSC-derived podocytes is provided here that was not described before in such detail.
As the overall protocol includes several different cell types, it is crucial to characterize the generated cell types at different steps. Cells are in culture for an extended period of time, so quality control should be performed at different passages. When working with hiPSCs, daily feeding as well as cell behavior and morphology monitoring is necessary. Sterility of the differentiation media must be ensured by filter sterilization through a 0.2 &#181;m filter. The whole protocol, from skin biopsy to hiPSC-derived podocytes, takes several months, but it is possible to freeze the cells at distinct stages of the process. Fibroblasts, selected hiPSC clones, and proliferative nephron progenitor cells after 7 days in nephron progenitor differentiation medium can be frozen, and a working cell bank can be generated.
Even though hiPSC-derived healthy podocytes develop a distinct network of primary and secondary foot processes (Figure 5A,B) and express&#160;typical podocyte-specific marker (Figure 6A-C), characteristic slit diaphragms, as seen in vivo, are hard to mimic in classical two-dimensional cell culture models. Moreover, intercell communication with other glomerular cell types is not possible in this mono-culture setting.
Due to their terminal differentiated state and lack of proliferation capacity, it is difficult to study podocytes ex vivo. With the help of conditionally immortalizing primary podocytes, it is possible to overcome this limitation by the insertion of a thermosensitive switch, resulting in a cell culture model where cells proliferate at 33 &#176;C and differentiate at 37 &#176;C13,14. Although these ciPodocytes have high potential for podocyte research, there are limitations, like the lack of marker expression, dedifferentiated morphology, and failure to form foot processes15,16.
The differentiation of podocytes from patient-derived somatic cells enables the generation and comparison of diseased podocytes with healthy control cells ex vivo. This enables us to study podocyte damage due to mutations in podocyte-specific genes. Furthermore, working with hiPSCs has the potential for creating three-dimensional cell culture disease models, or rather organoids43,44. Co-culture of hiPSC-derived podocytes with other glomerular cells, like glomerular endothelial cells or mesangial cells, might lead to new insights regarding intercell communication in health and glomerular disease.
Moreover, the characterization and treatment of the patient-specific podocytes can be performed ex vivo in high-throughput analysis. The individualized approach opens the opportunity to investigate novel therapeutic targets for specific mutations and to perform individualized medicine in the future.

Podocytes are specialized, post-mitotic renal epithelial cells that form the glomerular filtration barrier of the kidney along with the glomerular basement membrane (GBM), glomerular endothelial cells, and glycocalyx. Phenotypically, podocytes consist of a cell body and primary, microtubule-driven membrane extensions, as well as secondary extensions called foot processes1,2. The glomerular filtration barrier that filters urine from the blood is built of fenestrated endothelium, GBM, and a specialized type of intercellular junction that connects neighboring podocyte foot processes, called the slit diaphragm of podoctyes3. Under healthy conditions, proteins larger than albumin are retained from the filtration barrier due to their size and charge4.
Mutations in cytoskeletal- or podocyte-specific genes, as well as circulating factors affecting podocyte signaling pathways, are known to induce podocyte effacement, detachment, or apoptosis, resulting in proteinuria and glomerular sclerosis. In particular, cytoskeletal rearrangement, changes in podocyte polarity or damage of foot processes with an associated loss of slit junctions are&#160;pivotal5. Due to their terminally differentiated status, podocytes can hardly be replaced after detachment of the GBM. However, if podocytes are attached to the GBM, they can still recover from effacement and reform interdigitating foot processes6,7,8. Further understanding of the events that lead to podocyte damage in various glomerular disorders may provide novel therapeutic targets that will aid in developing treatments for these diseases. Podocyte damage is a hallmark of different glomerular diseases, including focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, minimal change disease, and membranous glomerulonephropathy, requiring reliable podocyte ex vivo models to study the pathological mechanisms and potential treatment approaches for these diseases9,10. Podocytes can be studied ex vivo by classical primary cell culture based on the isolation of glomeruli by differential sieving11. However, due to the terminally differentiated state with limited proliferation capacity, most researchers use mouse or human ciPodocyte cell lines that express temperature-sensitive variants of the SV40 large T antigen. Alternatively, ciPodocytes are isolated from transgenic mice harboring the SV40 Tag immortalizing gene1,12.
CiPodocytes proliferate at 33 &#176;C, but enter growth arrest and start differentiating at 37 &#176;C13,14. It has to be kept in mind that experimental data obtained with these cells should be interpreted with certain caution, as the cells are generated using an unnatural gene insertion15. As these cells harbor an immortalizing gene, the cellular physiology is altered because of ongoing proliferation12. Podocyte cell lines generated by this approach have recently been questioned, as mouse, human, and rat ciPodocytes express less than 5% of synaptopodin and nephrin at the protein level, as well as NPHS1 and NPHS2 at the mRNA level&#160;compared to glomerular expression16. Moreover, most podocyte cell lines do not express nephrin17,18. Chittiprol et al. also described a significant difference in cell motility and responses to puromycin and doxorubicin in ciPodocytes16. Podocytes can be found in urine after detachment from the GBM in different glomerular diseases19,20,21,22. Viable urinary podocytes can be cultivated ex vivo for up to 2-3 weeks, but most cells undergo apoptosis23,24. Interestingly, podocytes are not only found in the urine of patients with glomerular disease but also in the urine of healthy subjects, most likely when they are senescent-again with a limited potential of replication in culture24. Furthermore, the urinary-derived podocyte number is limited, and the cells dedifferentiate in culture, show less foot processes, change morphology, and most importantly have limited proliferation capacity. The expression of podocyte-specific genes is absent, disappears within a few weeks, or varies among these cell clones. Some cells positive for the podocyte-specific marker co-expressed the marker of tubular epithelial cells or myofibroblasts and mesangial cells, which suggests dedifferentiation and/or transdifferentiation of the cultured urinary podocytes24,25.
Recently, the generation of ciPodocyte cell lines derived from the urine of patients and healthy volunteers by transduction with a thermo-sensitive SV40 large T antigen and hTERT has been described26. mRNA expression for synaptopodin, nestin, and CD2-associated&#160;protein was detected, but podocin mRNA was absent in all clones. In addition to the problems with urinary podocytes, these cells also contain the inserted immortalizing gene, resulting in the disadvantages discussed above.
In contrast, human induced pluripotent stem cells (hiPSCs) have a huge capacity to self-renew and differentiate into multiple cell types under appropriate conditions. It has been shown before that hiPSCs can serve as an almost unlimited source of podocytes27,28.
Here, a two-step protocol to generate patient-specific podocytes from dermal fibroblasts of skin punch biopsies with subsequent episomal reprogramming into hiPSCs and a final differentiation into hiPSC-derived podocytes is described (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65364/65364fig01.jpg" /> 
Figure 1: Protocol to generate patient-specific hiPSC-derived podocytes. Graphical overview of the protocol to generate patient-specific podocytes from dermal fibroblasts of a skin biopsy by reprogramming into hiPSCs and differentiation into podocytes. <a href="https://www.jove.com/files/ftp_upload/65364/65364fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
As a first step, somatic dermal fibroblasts were outgrown from a skin punch biopsy and reprogrammed into hiPSCs using an integration-free method by electroporation with plasmids expressing the transcription factors OCT3/4, KLF4, SOX2, and c-MYC29,30,31. Arising hiPSC colonies were subsequently selected and expanded. Differentiation began with the induction of the mesodermal lineage by activation of the WNT signaling pathway, followed by the generation of nephron progenitor cells that were still able to proliferate. Finally, the cells were differentiated into podocytes. In this procedure, we modified and combined previously published protocols for episomal reprogramming for the generation of hiPSCs by Bang et al.32 and Okita et al.33, as well as a protocol for the differentiation of hiPSCs into podocytes by Musah et al.28,34,35.
Indeed, podocytes generated by our protocol had a phenotype closer to podocytes in vivo, regarding the development of a distinct network of primary and secondary foot processes and the expression of podocyte-specific markers, like synaptopodin, podocin, and nephrin. With the use of hiPSC-derived podocytes, the patient&#39;s genetic background is maintained during reprogramming and differentiation. This enables patient-specific podocyte disease modelling and the discovery of potential therapeutic substances ex vivo in an almost unlimited cell number. Moreover, this protocol is minimally invasive, cost-efficient, ethically acceptable and may facilitate new avenues for drug development.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 &#181;m sterile filter</td>
			<td>Rotilab</td>
			<td>P668.1</td>
			<td>for sterilization of differentiation medium</td>
		</tr>
		<tr>
			<td>all-trans retinoic acid</td>
			<td>Stem Cell Technologies</td>
			<td>72262</td>
			<td>supplement for differentiation</td>
		</tr>
		<tr>
			<td>B27 supplement (50 x), serum free</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td>supplement for serum-free differentiation medium</td>
		</tr>
		<tr>
			<td>BG iMatrix-511 Silk</td>
			<td>biogems</td>
			<td>RL511S</td>
			<td>additional option of extracellular matrix reagent used in coating solution to coat cell culture plastics suitable for hiPSC culture</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)&#160;</td>
			<td>Roth</td>
			<td>8076.4</td>
			<td></td>
		</tr>
		<tr>
			<td>CELLSTAR Filter Cap Cell Culture Flasks, T75, 250 mL</td>
			<td>Greiner bio-one</td>
			<td>82050-856</td>
			<td>cell culture plastics suitable for fibroblast culture</td>
		</tr>
		<tr>
			<td>CHIR99021 (5 mg)</td>
			<td>Sigma-Aldrich</td>
			<td>252917-06-9&#160;</td>
			<td>supplement for differentiation</td>
		</tr>
		<tr>
			<td>Corning Matrigel hESC qualified matrix&#160;</td>
			<td>Corning</td>
			<td>354277</td>
			<td>additional option of extracellular matrix reagent used in coating solution to coat cell culture plastics suitable for hiPSC culture (solubilized basement membrane matrix)</td>
		</tr>
		<tr>
			<td>countess Cell Counting Chamber Slides</td>
			<td>Invitrogen</td>
			<td>C10283</td>
			<td>to count cells</td>
		</tr>
		<tr>
			<td>countess II FL&#160; Automated Cell Counter</td>
			<td>Invitrogen</td>
			<td></td>
			<td>to count cells</td>
		</tr>
		<tr>
			<td>cryoPure tubes, 2 ml, QuickSeal screw cap, white</td>
			<td>Sarstedt</td>
			<td>72380</td>
			<td>cryovials for freezing of cells</td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide (DMSO)&#160;</td>
			<td>Roth</td>
			<td>A994.1</td>
			<td>for fibroblast freezing medium</td>
		</tr>
		<tr>
			<td>DMEM/F12 (1:1) (1 x)</td>
			<td>Gibco</td>
			<td>11320074</td>
			<td>basic medium for differentiation</td>
		</tr>
		<tr>
			<td>DMEM/F12 + Glutamax</td>
			<td>Gibco</td>
			<td>10565018</td>
			<td>basic medium for fibroblast medium</td>
		</tr>
		<tr>
			<td>EVOS M5000 Imaging System</td>
			<td>Thermo Fisher Scientific</td>
			<td>AMF5000</td>
			<td>phase contrast microscope</td>
		</tr>
		<tr>
			<td>fetal bovine serum premium, inactivated (FCS)</td>
			<td>PAN Biotech</td>
			<td>P301902</td>
			<td>serum for fibroblast medium, fibroblast freezing medium and podocyte maintenance medium</td>
		</tr>
		<tr>
			<td>fisherbrand Electroporation Cuvettes Plus, 4 mm gap, 800 &#181;L capacity, sterile</td>
			<td>Fisherbrand</td>
			<td>FB104</td>
			<td>cuvette used for electroporation/episomal reprogramming of fibroblasts (4mm gap)</td>
		</tr>
		<tr>
			<td>fluoromount-G Mounting Medium, with DAPI</td>
			<td>Invitrogen</td>
			<td>00-4959-52</td>
			<td>mounting medium containing dapi</td>
		</tr>
		<tr>
			<td>gauge needle (0.6 x 30 mm)</td>
			<td>BD Microlance3</td>
			<td>300700</td>
			<td>for separation of hiPSC colonies into small pieces</td>
		</tr>
		<tr>
			<td>human Recombinant Activin A Protein</td>
			<td>78001.1</td>
			<td>Stem cell technologies</td>
			<td>supplement for differentiation</td>
		</tr>
		<tr>
			<td>human recombinant bone morphogenetic protein 7 (BMP7)</td>
			<td>Peprotech</td>
			<td>120-03P</td>
			<td>supplement for differentiation</td>
		</tr>
		<tr>
			<td>human VEGF-165 Recombinant Protein</td>
			<td>Thermo Scientific</td>
			<td>PHC9394</td>
			<td>supplement for differentiation</td>
		</tr>
		<tr>
			<td>insulin-transferrin-selenium (ITS -G) (100 x)</td>
			<td>Gibco</td>
			<td>41400045</td>
			<td>supplement for podocyte maintenance medium</td>
		</tr>
		<tr>
			<td>LB medium</td>
			<td>Roth</td>
			<td>X964.1</td>
			<td>for sterility test of hiPSC culture</td>
		</tr>
		<tr>
			<td>lookOut Mycoplasma PCR Detection Kit</td>
			<td>Sigma Aldrich</td>
			<td>MP0035-1KT</td>
			<td>commercial mycoplasma detection kit</td>
		</tr>
		<tr>
			<td>microscope slides</td>
			<td>Diagonal GmbH &#38; Co.KG</td>
			<td>21,102</td>
			<td></td>
		</tr>
		<tr>
			<td>microtube 1.5 mL&#160;</td>
			<td>Sarstedt</td>
			<td>72706400</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1 Complete Kit</td>
			<td>Stem Cell Technologies</td>
			<td>85850</td>
			<td>basic medium for serum-free hiPSC culture medium</td>
		</tr>
		<tr>
			<td>nalgene freezing container Mr.Frosty</td>
			<td>Roth</td>
			<td>AC96.1&#160;</td>
			<td>to ensure optimal freezing conditions</td>
		</tr>
		<tr>
			<td>normal goat serum</td>
			<td>abcam</td>
			<td>ab 7481</td>
			<td>for preincubation solution and antibody diluent</td>
		</tr>
		<tr>
			<td>nunc 24 well plates</td>
			<td>Thermo Scientific</td>
			<td>142485</td>
			<td>cell culture plastics suitable for hiPSC culture</td>
		</tr>
		<tr>
			<td>nunc 48 well plates&#160;</td>
			<td>Thermo Scientific</td>
			<td>152640</td>
			<td>cell culture plastics suitable for hiPSC culture</td>
		</tr>
		<tr>
			<td>nunc 6 well plates&#160;</td>
			<td>Thermo Scientific</td>
			<td>140685</td>
			<td>cell culture plastics suitable for hiPSC culture</td>
		</tr>
		<tr>
			<td>nunc EasYDish Dishes 100 mm</td>
			<td>Thermo Scientific</td>
			<td>150466</td>
			<td>cell culture plastics suitable for hiPSC culture</td>
		</tr>
		<tr>
			<td>nunc MicroWell 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate</td>
			<td>Thermo Scientific</td>
			<td>167008</td>
			<td>cell culture plastics suitable for hiPSC culture</td>
		</tr>
		<tr>
			<td>nutriFreez D10 Cryopreservation Medium</td>
			<td>Sartorius</td>
			<td>05-713-1E&#160;</td>
			<td>serum-free cryopreservation medium for cryopreservation of hiPSC and nephron progenitor cells</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Gibco</td>
			<td>11058021</td>
			<td>electroporation medium&#160;</td>
		</tr>
		<tr>
			<td>pCXLE-hMLN</td>
			<td>Addgene</td>
			<td>#27079</td>
			<td>plasmid for episomal reprogramming</td>
		</tr>
		<tr>
			<td>pCXLE-hOCT3/4 plasmid</td>
			<td>Addgene</td>
			<td>#27077</td>
			<td>plasmid for episomal reprogramming</td>
		</tr>
		<tr>
			<td>pCXLE-hSK plasmid</td>
			<td>Addgene</td>
			<td>#27078</td>
			<td>plasmid for episomal reprogramming</td>
		</tr>
		<tr>
			<td>penicillin-streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333-100ML</td>
			<td>to avoid bacterial contamination</td>
		</tr>
		<tr>
			<td>plastic coverslips</td>
			<td>Sarstedt</td>
			<td>&#160; 83.1840.002</td>
			<td>for immunofluorescent stainings of hiPSCs and hiPSC-derived podocytes</td>
		</tr>
		<tr>
			<td>ROTI Histofix</td>
			<td>Roth</td>
			<td>P087.3</td>
			<td>commercial paraformaldehyde (4 %) for fixation of cells</td>
		</tr>
		<tr>
			<td>RPMI 1640 + L-Glutamine</td>
			<td>Gibco</td>
			<td>21875034</td>
			<td>basic medium for podocyte maintenance medium</td>
		</tr>
		<tr>
			<td>staining chamber StainTray Black lid</td>
			<td>Roth</td>
			<td>HA51.1</td>
			<td></td>
		</tr>
		<tr>
			<td>stemPro Accutase Cell Dissociation Reagent</td>
			<td>Gibco</td>
			<td>A1110501</td>
			<td>enzymatic cell detachment solution used for dissociation of hiPSCs</td>
		</tr>
		<tr>
			<td>sterile phosphate buffered saline (PBS) (1 x)</td>
			<td>Gibco</td>
			<td>14190094</td>
			<td>used for washing and coating</td>
		</tr>
		<tr>
			<td>sterile water</td>
			<td>Roth</td>
			<td>T1432</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe without needle 20 mL</td>
			<td>BD Plastipak</td>
			<td>300629</td>
			<td>to filter sterilize differentiation medium</td>
		</tr>
		<tr>
			<td>TC dish 100 mm</td>
			<td>Sarstedt</td>
			<td>8,33,902</td>
			<td>sterile cell culture plastics used for cutting the skin biopsy and fibroblast culture</td>
		</tr>
		<tr>
			<td>TC dish 35 mm</td>
			<td>Sarstedt</td>
			<td>8,33,900</td>
			<td>sterile cell culture plastics used for outgrowing fibroblasts from skin biopsy</td>
		</tr>
		<tr>
			<td>triton X 100</td>
			<td>Roth</td>
			<td>3051.3</td>
			<td>for preincubation solution&#160;</td>
		</tr>
		<tr>
			<td>trypan Blue Stain (0.4 %) for use with the Countess Automated Cell Counter</td>
			<td>Invitrogen</td>
			<td>T10282</td>
			<td>to count cells</td>
		</tr>
		<tr>
			<td>trypsin-EDTA (10 x)&#160;</td>
			<td>Biowest</td>
			<td>X0930-100</td>
			<td>dissociation reagent used for fibroblasts and nephron progenitor cells</td>
		</tr>
		<tr>
			<td>tube 15 mL</td>
			<td>Greiner bio-one</td>
			<td>188271-N</td>
			<td></td>
		</tr>
		<tr>
			<td>tube 50 mL</td>
			<td>Greiner bio-one</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>vitronectin ACF</td>
			<td>Sartorius</td>
			<td>05-754-0002</td>
			<td>extracellular matrix reagent used in coating solution to coat cell culture plastics suitable for hiPSC culture</td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride (10 mg)</td>
			<td>Tocris</td>
			<td>1254</td>
			<td>to avoid apoptosis of hiPSCs during splitting</td>
		</tr>
		<tr>
			<td>Primary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OCT4&#160;</td>
			<td>Stem Cell Technologies</td>
			<td>60093.1</td>
			<td>pluripotency marker, dilution 1:200</td>
		</tr>
		<tr>
			<td>SSEA-4</td>
			<td>Stem Cell Technologies</td>
			<td>60062FI.1</td>
			<td>pluripotency marker, dilution 1:100</td>
		</tr>
		<tr>
			<td>Ki67</td>
			<td>Abcam</td>
			<td>ab15580</td>
			<td>proliferation marker, dilution 1:300</td>
		</tr>
		<tr>
			<td>synaptopodin</td>
			<td>Proteintech</td>
			<td>21064-1-AP</td>
			<td>podocyte-specific marker, dilution 1:200</td>
		</tr>
		<tr>
			<td>nephrin</td>
			<td>Progen</td>
			<td>GP-N2</td>
			<td>podocyte-specific marker, dilution 1:25</td>
		</tr>
		<tr>
			<td>podocin</td>
			<td>proteintech</td>
			<td>20384-1-AP</td>
			<td>podocyte-specific marker, dilution 1:100</td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti-Guinea Pig IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555</td>
			<td>Invitrogen</td>
			<td>A21435</td>
			<td>secondary anditbody, dilution 1:1000</td>
		</tr>
		<tr>
			<td>alexa Fluor 647 Goat Anti-Rabbit SFX Kit, highly cross-adsorbed</td>
			<td>Invitrogen</td>
			<td>A31634</td>
			<td>secondary anditbody, dilution 1:1000</td>
		</tr>
		<tr>
			<td>donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A21206</td>
			<td>secondary anditbody, dilution 1:1000</td>
		</tr>
		<tr>
			<td>goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 555</td>
			<td>Invitrogen</td>
			<td>A21422</td>
			<td>secondary anditbody, dilution 1:1000</td>
		</tr>
		<tr>
			<td>goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A11001</td>
			<td>secondary anditbody, dilution 1:1000</td>
		</tr>
	</tbody>
</table>

generation of patient-derived podocytes from skin biopsies

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the glomerulus. Mutations in podocyte-specific genes can cause focal segmental glomerulosclerosis (FSGS), and podocytes are also affected in many other primary and secondary nephropathies. Due to their differentiated nature, primary cell culture models are limited for podocytes.&#160;Therefore, commonly conditionally immortalized cells are used. However, these conditionally immortalized podocytes (ciPodocytes) have several limitations: the cells can dedifferentiate in culture, especially when they reach confluency, and several podocyte-specific markers are either only slightly or not expressed at all. This brings the use of ciPodocytes and their applicability for physiological, pathophysiological, and clinical reach into question. Here, we describe a protocol for the generation of human podocytes-including patient-specific podocytes-from a skin punch biopsy by episomal reprogramming of dermal fibroblasts into hiPSCs and subsequent differentiation into podocytes. These podocytes resemble in vivo podocytes much better in terms of morphological characteristics, like the development of foot processes and the expression of the podocyte-specific marker. Finally, yet importantly, these cells maintain patients&#39; mutations, resulting in an improved ex vivo model to study podocyte diseases and potential therapeutic substances in an individualized approach.

Author Spotlight: Generation of Patient-Derived Podocytes from Skin Biopsies  

Generation of Patient-Derived Podocytes from Skin Biopsies

We describe a protocol for generating human podocytes by episomal reprogramming dermal fibroblasts into human-induced pluripotent stem cells. These cells resemble in vivo podocytes much better than immortalized podocytes in terms of podocyte food processes and expression of podocyte specific marker. Podocytes are terminally differentiated cells.Therefore, these cells no longer proliferate and primary podocyte analysis in cell culture is limited. Even though the problem of limited cell number was overcome by using conditionally immortalized podocytes, these podocytes present a dedifferentiated phenotype and behavior, which questions their use in basic research. With the help of induced pluripotent stem cells, podocytes can be generated in vitro in an almost unlimited cell number with typical podocyte morphology including distinct food processes and expression of podocyte specific marker.When using the patient-specific stem cells to differentiate into podocytes, it is possible to study the cell's disease-specific alterations ex vivo. In case, the initial skin biopsy was taken from a patient with a genetic podocyte disease. Podocytes generated with our protocol are personalized diseased cells carrying the patient's mutation.Thus, these cells represent an improved ex vivo model to study podocyte diseases and potential therapeutic substances in an individualized approach.

With this step-by-step protocol that combines episomal reprogramming and differentiation, it is possible to generate podocytes carrying a patient&#39;s mutation in a podocyte-relevant gene. This enables the analysis of disease-specific podocyte alterations ex vivo. The patient&#39;s genetic background is preserved during the protocol during all the different cell stages. In addition, the limitation of insufficient cell number of terminally differentiated non-proliferating podocytes can be overcome by using hiPSC-derived podocytes. Although it takes several months until hiPSC-podocytes are generated from outgrown dermal fibroblasts via reprogramming into hiPSCs and subsequent differentiation into podocytes, it is possible to freeze cells at three different steps of the protocol (Figure 2). Freezing is possible for fibroblasts, hiPSCs, and nephron progenitor cells after differentiation in nephron progenitor differentiation medium for 7 days. Therefore, the generation of working cell banks and large-scale experiments is possible.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65364/65364fig02.jpg" /> 
Figure 2: Individual steps of the protocol through a phase contrast microscope.&#160;(A) Outgrown dermal fibroblasts. (B) hiPSC colony formation after reprogramming. (C) Selected hiPSC culture. (D) Mesoderm cells after 2 days of differentiation. (E) Nephron progenitor cells after 14 days of differentiation in nephron progenitor differentiation medium. (F) Terminal differentiated hiPSC-derived podocytes. Scale bars represent 300 &#181;m (A,B,D-F) and 150 &#181;m (C). The snowflake highlights possible freezing steps. <a href="https://www.jove.com/files/ftp_upload/65364/65364fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
As the generated hiPSC-derived podocytes traverse a long protocol with drastic changes regarding cell type and morphology, cell characterization is obligatory. Cell morphology, like size and cell shape, as well as growth behavior, can be monitored using a phase contrast microscope. Fibroblasts present a long spindle-like phenotype with a size of 150 &#181;m to 300 &#181;m&#160;(Figure 2A). After reprogramming, colonies with 50 &#181;m&#160;small hiPSCs occur. These colonies display distinct borders and hiPSCs distinguished with a high nuclei-to-body ratio and increased proliferation rate (Figure 2C). Since podocytes are terminally differentiated cells, the proliferation rate decreases with progressive differentiation, and the cell morphology changes to 300 &#181;m&#160;large star-shaped podocytes with prominent foot processes (Figure 2F).
Confluency is a critical point of hiPSC culture, and spontaneous differentiation can appear when colonies grow too dense (Figure 3). These colonies must be removed before passaging by scraping the affected parts of the culture dish.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65364/65364fig04.jpg" /> 
Figure 3: Examples of hiPSCs of different quality. Phase contrast images of successfully (A) reprogrammed hiPSC colonies and (B) selected hiPSCs, as well as (C) spontaneous differentiation, (D,E) non-hiPSC colonies, and (F) a too-dense hiPSC culture. Scale bars represent 300 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65364/65364fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The different cell types of this protocol must be validated regarding the expression of a specific marker. After reprogramming, hiPSCs regain pluripotency capacity and express proliferation and pluripotency markers like SSEA4, OCT3/4, and Ki67 (Figure 4)38,39,40. It is known that reprogramming, as well as the lengthy culture of hiPSCs and differentiation, can lead to an abnormal karyotype, or rather induce mutations41. Therefore, the genetic background of the cultured cells should be monitored over time and at different passages by g-banding and whole exome sequencing.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65364/65364fig03.jpg" /> 
Figure 4: Characterization of generated hiPSCs by immunofluorescence staining. Characterization of generated hiPSCs by staining for (A) the proliferative marker Ki67, as well as the pluripotency markers (B) OCT3/4 and (C) SSEA4. Scale bars represent 100 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65364/65364fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Morphologically, hiPSC-derived podocytes appear star-shaped and express a distinct network of long primary and secondary foot processes compared to ciPodocytes (Figure 5). HiPSC-derived podocytes express podocyte-specific marker proteins like synaptopodin, nephrin, and podocin (Figure 6A-C)42.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65364/65364fig05.jpg" /> 
Figure 5: Morphology of hiPSC-derived podocytes compared to ciPodocytes.&#160;Comparison of (A,B) hiPSC-derived podocytes from a healthy donor to (C,D) ciPodocytes regarding cell morphology and filopodia by using a phase contrast microscope (A,C) and scanning electron microscope (B,D). Scale bars represent 150 &#181;m (A,C) and 20 &#181;m (B,D). <a href="https://www.jove.com/files/ftp_upload/65364/65364fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Therefore, the comparison of hiPSC-derived podocytes not only from healthy donors, but also from patients with mutations in podocyte-specific genes, allows for individualized characterization of the patient&#39;s mutation ex vivo.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65364/65364fig06.jpg" /> 
Figure 6: Characterization of a podocyte-specific marker in hiPSC-derived podocytes and ciPodocytes. Comparison of (A-C) hiPSC-derived podocytes from a healthy donor to (D-F) ciPodocytes regarding podocyte-specific marker proteins synaptopodin (A,D), nephrin (B,E), and podocin (C,F). Scale bars represent 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65364/65364fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1. Composition of all cell culture media and solutions used in the study. <a href="https://www.jove.com/files/ftp_upload/65364/Table 1_REv2.zip" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Generation of Patient-Derived Podocytes from Skin Biopsies at JoVE.com

This manuscript describes a two-step protocol to generate patient-specific podocytes from dermal fibroblasts via episomal reprogramming into human-induced pluripotent stem cells (hiPSCs) and subsequent differentiation into podocytes.

Podocytes are epithelial cells sitting on the urinary site of the glomerular filtration barrier that contribute to the selective filter function of the ...

generation-of-patient-derived-podocytes-from-skin-biopsies

Research

JoVE Journal

Biology

﻿Outgrowth of Dermal Fibroblasts from a Skin Punch Biopsy

Outgrowth of Dermal Fibroblasts from a Skin Punch Biopsy  

To begin, take the skin biopsy tube with fibroblast medium. Remove the medium, and wash the skin punch biopsy thrice with five milliliters of sterile, pre-warmed PBS. Using sterile forceps, transfer the skin punch biopsy to a 10-centimeter cell culture plate, and cut it widthwise into three or four pieces with a sterile scalpel, leaving the epidermis and dermis.Transfer each piece into a sterile, 35-millimeter, plastic cell culture dish, and press it gently into the culture dish. Allow it to dry for five to 10 minutes until the liquid evaporates and the biopsy is attached to the cell culture plastic. Using a 1, 000-microliter pipette, add one milliliter of fibroblast medium dropwise around the biopsy to a final volume of three milliliters.Incubate the sample for seven days at 37 degrees Celsius and 5%carbon dioxide without feeding or moving the dish. At the end of the incubation, change the medium to fresh, pre-warmed fibroblast medium. Finally, use a phase contrast microscope to monitor the outgrown spindle-like fibroblasts around the skin biopsy, and freeze the selected cells after growing them.

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (hiPSCs)

Episomal Reprogramming of Dermal Fibroblasts to Generate Human-Induced Pluripotent Stem Cells (HiPSCs)  

To begin, take a flask of cultured fibroblast cells, coat a 10-centimeter cell culture plate suitable for hiPSC culture with four milliliters of cold coating solution per plate, and incubate the plate for one hour at 37 degrees Celsius. Afterward, remove the media from the fibroblasts and wash them with four milliliters of pre-warmed PBS per flask. Aspirate the PBS and associate the fibroblasts with four milliliters of Trypsin-EDTA per flask by incubating for five to seven minutes at 37 degrees Celsius.Monitor cell detachment using a phase contrast microscope, and if needed, tap against the side of the flask to detach the cells from the plastic surface. Transfer the detached cells to a 50-milliliter conical tube and wash the empty flasks with six milliliters of pre-warmed fibroblast medium. Collect and pool the remaining cells in the conical tube.Then centrifuge the cells at 200 g for five minutes at 20 degrees Celsius. Aspirate the supernatant and re-suspend the cell pellet in three milliliters of fresh, pre-warmed PBS. Count the cells, transfer 1.5 times 10 to the sixth fibroblasts into a new conical tube and fill up with PBS to the five millimeter mark.Centrifuge the tube at 200 g for five minutes at 20 degrees Celsius. Discard the supernatant, re-suspend the cell pellet in five milliliters of electroporation medium, and centrifuge again. In the meantime, aspirate the coating solution from the coated 10-centimeter cell culture plate and add seven milliliters of pre-warmed fibroblast medium.At the end of the centrifugation, discard the supernatant and re-suspend the cell pellet in electroporation medium with a concentration of 1.5 times 10 to the sixth cells in 250 microliters. Transfer the 250 microliter cell suspension to an electroporation cuvette with a four millimeter gap distance. Next, prepare a plasma transfection mix by adding four micrograms of each plasmid to a total volume of 50 microliters of electroporation medium.Transfer the transfection mix to the cuvette, mix gently by flicking, and electroporate with one pulse at 280 volts. Finally, transfer 150 microliters of the electroporated fibroblasts to the prepared 10-centimeter cell culture plate. Agitate the plate thrice in all directions to distribute the cells, and incubate overnight.Fibroblasts present in a long, spindle-like phenotype, the size of 150 to 300 nanometers. After reprogramming, colonies with 50 micrometer small hiPSCs occur displaying distinct borders. The hiPSCs are distinguished by a high nuclei to body ratio and increased proliferation rate.

Selection, Expansion, and Quality Control of hiPSCs Generated from Dermal Fibroblasts

To begin, take the plate of electroporated fibroblasts and observe the formation of hiPSC colonies under a 10x to 20x objective phase contrast microscope. The hiPSC colonies with 300 micrometer diameter, distinct borders, and a high nuclei to body ratio are suitable for picking. Before picking, coat the wells of a 96 well plate suitable for hiPSC culture with 100 microliters of coating solution.And incubate for one hour at 37 degrees Celsius. During incubation, mark the colonies of interest at the bottom of the cell culture dish with a pen. For the final preparation of the 96 well plate, remove the coating solution and add 100 microliters of pre-warmed hiPSC culture medium, containing 10 micromoles of ROCK inhibitor Y-27632 to each well.Next, wash the cells with pre-warmed PBS and add fresh pre-warmed hiPSC culture medium containing 10 micromoles of ROCK inhibitor Y-27632 before picking to remove dead cells. Pick the hiPSC colonies using a gauge needle and divide the colonies into small pieces by drawing a grid into each colony. Afterward, use a phase contrast microscope to check that the colonies are successfully divided into pieces.Finally, transfer the colonies with a 100 microliter pipette to the prepared 96 well plate, holding the pipette upright over the colony. Allow the cells to attach overnight at 37 degrees Celsius and 5%carbon dioxide without disturbance. The following day, replace the medium with 200 microliters of fresh hiPSC culture medium to remove the ROCK inhibitor Y-27632 and place the plates back in the incubator.Monitor the 96 well plate and mark the wells with successfully picked clones. The marked clones can be expanded and frozen at this point. Phase contrast images have successfully reprogrammed hiPSC colonies, selected hiPSCs, spontaneously differentiated colonies, non-hiPSC colonies, and a too dense hiPSC culture are presented.

973 Views.  Friedrich-Alexander University (FAU) Erlangen-Nürnberg.  This manuscript describes a two-step protocol to generate patient-specific podocytes from dermal fibroblasts via episomal reprogramming into human-induced pluripotent stem cells (hiPSCs) and subsequent differentiation into podocytes.

Using Skin Biopsies to Generate Patient-Derived Podocytes

Summary