This video demonstrates a protocol for culturing fibroblasts from a skin biopsy. It details the steps of preparing the biopsy, transferring it to a culture dish, incubating in specific conditions, changing the medium, and monitoring the growth of fibroblasts using a phase contrast microscope before freezing selected cells.

﻿outgrowth of dermal fibroblasts from a skin punch biopsy

Using Skin Biopsies to Generate Patient-Derived Podocytes

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.

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.

Watch this Scientific Journal Video about ﻿Outgrowth of Dermal Fibroblasts from a Skin Punch Biopsy at JoVE.com

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.

outgrowth-of-dermal-fibroblasts-from-a-skin-punch-biopsy

Research

JoVE Journal

Biology

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.

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 ...

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.