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.

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

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

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.

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 ...

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1.9K 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.

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

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.

Here, we describe a protocol based on epigenetic reprogramming of human embryonic stem cells (hESCs) toward generating a homogeneous population of skeletal muscle progenitors that under permissive culture conditions form three-dimensional clusters of contractile myofibers (myospheres), which recapitulate biological features of human skeletal muscles.

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Quantifying Leukocyte Egress via Lymphatic Vessels from Murine Skin and Tumors

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the resolution of peripheral tissue responses. While a variety of methods are used to quantify leukocyte egress from non-lymphoid, peripheral tissues, the cellular and molecular mechanisms that govern context-dependent egress remain poorly understood. Here, we describe the use of in situ photoconversion for quantitative analysis of leukocyte egress from murine skin and tumors. Photoconversion allows for the direct labeling of leukocytes resident within cutaneous tissue. Though skin exposure to violet light induces local inflammatory responses characterized by leukocyte infiltrates and vascular leakiness, in a head-to-head comparison with transdermal application of fluorescent tracers, photoconversion specifically labeled migratory dendritic cell populations and simultaneously enabled the quantification of myeloid and lymphoid egress from cutaneous microenvironments and tumors. The mechanisms of leukocyte egress remain a missing component in our understanding of intratumoral leukocyte complexity, and thus the application of the tools described herein will provide unique insight into the dynamics of tumor immune microenvironments both at steady state and in response to therapy.

Here, we demonstrate the methods for in vivo quantification of leukocyte egress from na&#239;ve, inflamed, and malignant murine skin. We perform a head-to-head comparison of two models: transdermal FITC application and in situ photoconversion. Furthermore, we demonstrate the utility of photoconversion for tracking leukocyte egress from cutaneous tumors.

Leukocyte egress from peripheral tissues to draining lymph nodes is not only critical for immune surveillance and initiation but also contributes to the ...

Immunolabelling Myofiber Degeneration in Muscle Biopsies

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. Therapeutic options addressing the causes of muscular dystrophy pathogenesis are expected to alleviate muscle degeneration. Therefore, a method to assay and quantify the extent of cell death in muscle biopsies is needed. Conventional methods to observe myofiber degeneration in situ are either poorly quantitative or rely on the injection of vital dyes. In this article, an immunofluorescence protocol is described that stains necrotic myofibers by targeting immunoglobulin G (IgG) uptake by myofibers. The IgG uptake method is based on cell features characterizing the necrotic demise, including 1) the loss of plasma membrane integrity with the release of damage-associated molecular patterns and 2) the uptake of plasmatic proteins. In murine cross-sections, the co-immunolabelling of myofibers, extracellular matrix proteins, and mouse IgG allows clean and straightforward identification of myofibers with necrotic fate. This simple method is suitable for quantitative analysis and applicable to all species, including human samples, and does not require the injection of vital dye. The staining of necrotic myofibers by IgG uptake can also be paired with other co-immunolabelling.

Described here is a protocol for direct immunolabelling of necrotic myofibers in muscle cryosections. Necrotic cells are permeable to serum proteins, including immunoglobulin G (IgG). Revealing the uptake of IgG by myofibers allows the identification and quantification of myofibers that undergo necrosis regardless of muscle condition.

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. ...

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-P&#233;rot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Presented here is a protocol to assess the contractile properties of striated muscle myofibrils with nano-Newton resolution. The protocol employs a setup with an interferometry-based, optical force probe. This setup generates data with a high signal-to-noise ratio and enables the assessment of the contractile kinetics of myofibrils.

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of ...

Y-27632 Enriches the Yield of Human Melanocytes from Adult Skin Tissues

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

This paper reports that the addition of Y-27632 to TIVA medium can significantly increase the yield of melanocytes from adult skin tissues.

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical ...

Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle biopsies are often collected for these endeavors. However, relatively few technical descriptions of biopsy procedures, outside of the commonly used musculus vastus lateralis, are available. Although the biopsy techniques are often adjusted to accommodate the characteristics of each muscle under study, few technical reports share these changes to the greater community. Thus, muscle tissue from human participants is often wasted as the operator reinvents the wheel. Expanding the available material on biopsies from a variety of muscles can reduce the incident of failed biopsies. This technical report describes a variation of the modified Bergstr&#246;m technique on the musculus tibialis anterior that limits fiber damage and provides fiber lengths adequate for mechanical evaluation. The surgery is an outpatient procedure that can be completed in an hour. The recovery period for this procedure is immediate for light activity (i.e., walking), up to three days for the resumption of normal physical activity, and about one week for wound care. The extracted tissue can be used for mechanical force experiments and here we present representative activation data. This protocol is appropriate for most collection purposes, potentially adaptable to other skeletal muscles, and may be improved by modifications to the collection needle.

This technical report describes a variation of the modified Bergstr&#246;m technique for the biopsy of the musculus tibialis anterior that limits fiber damage.

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle ...