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

피부 생검을 사용하여 환자 유래 족세포(Patient-derived Podocyte) 생성

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.

저자 스포트라이트: Generation of Patient-Derived Podocytes from Skin Biopsyes  

피부 생검에서 환자 유래 Podocytes의 생성

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  

Skin Punch Biopsy에서 피부 섬유아세포의 성장

시작하려면 섬유아세포 배지가 있는 피부 생검 튜브를 채취합니다. 배지를 제거하고, 피부 펀치 생검을 5밀리리터의 멸균되고 예열된 PBS로 3회 세척한다. 멸균 겸자를 사용하여 피부 펀치 생검을 10cm 세포 배양 플레이트에 옮기고 멸균 메스로 너비로 3-4 개로 자르고 표피와 진피를 남깁니다.각 조각을 멸균된 35mm 플라스틱 세포 배양 접시에 옮기고 배양 접시에 부드럽게 누릅니다. 액체가 증발하고 생검이 세포 배양 플라스틱에 부착될 때까지 5-10분 동안 건조시킵니다. 1, 000 마이크로 리터 피펫을 사용하여 생검 주위에 1 밀리리터의 섬유 아세포 배지를 3 밀리리터의 최종 부피에 적가합니다.접시를 먹이거나 이동하지 않고 섭씨 37도와 이산화탄소 5%에서 7일 동안 샘플을 배양합니다. 배양이 끝나면 배지를 신선하고 예열된 섬유아세포 배지로 변경합니다. 마지막으로, 위상차 현미경을 사용하여 피부 생검 주변의 자란 방추형 섬유아세포를 모니터링하고 선택한 세포를 성장시킨 후 동결합니다.

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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 ...

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생물학

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)  

인간 유도 만능 줄기 세포(hiPSC)를 생성하기 위한 진피 섬유아세포의 에피솜 재프로그래밍

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

진피 섬유아세포에서 생성된 hiPSC의 선택, 확장 및 품질 관리