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

皮膚生検を使用して患者由来の足細胞を生成する

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.

著者スポットライト:皮膚生検からの患者由来足細胞の生成  

皮膚生検からの患者由来足細胞の生成

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  

皮膚パンチ生検からの皮膚線維芽細胞の増殖

はじめに、線維芽細胞培地で皮膚生検チューブを取ります。培地を取り除き、5ミリリットルの滅菌済み、予熱したPBSで皮膚パンチ生検を3回洗浄します。滅菌鉗子を用いて、皮膚パンチ生検を10cmの細胞培養プレートに移し、滅菌メスで幅方向に3〜4個に切断し、表皮と真皮を残します。各ピースを滅菌済みの35ミリメートルのプラスチック製細胞培養皿に移し、培養皿に静かに押し込みます。液体が蒸発し、生検が細胞培養プラスチックに付着するまで、5〜10分間乾燥させます。1, 000マイクロリットルのピペットを使用して、生検の周りに1ミリリットルの線維芽細胞培地を滴下して3ミリリットルの最終容量まで加えます。皿に餌を与えたり動かしたりせずに、摂氏37度と5%の二酸化炭素で7日間サンプルをインキュベートします。インキュベーションの最後に、培地を新鮮で事前に温めた線維芽細胞培地に変更します。最後に、位相差顕微鏡を使用して、皮膚生検の周りの成長した紡錘体様線維芽細胞を監視し、選択した細胞を成長させた後に凍結します。

Research

JoVE Journal

Biology

﻿Outgrowth of Dermal Fibroblasts from a Skin Punch Biopsy

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 ...