Generation and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine

摘要

The present protocol describes a method to isolate, expand, and reprogram human and non-human primate urine-derived cells to induced pluripotent stem cells (iPSCs), as well as instructions for feeder-free maintenance of the newly generated iPSCs.

摘要

Cross-species approaches studying primate pluripotent stem cells and their derivatives are crucial to better understand the molecular and cellular mechanisms of disease, development, and evolution. To make primate induced pluripotent stem cells (iPSCs) more accessible, this paper presents a non-invasive method to generate human and non-human primate iPSCs from urine-derived cells, and their maintenance using a feeder-free culturing method.

The urine can be sampled from a non-sterile environment (e.g., the cage of the animal) and treated with a broad-spectrum antibiotic cocktail during primary cell culture to reduce contamination efficiently. After propagation of the urine-derived cells, iPSCs are generated by a modified transduction method of a commercially available Sendai virus vector system. First iPSC colonies may already be visible after 5 days, and can be picked after 10 days at the earliest. Routine clump passaging with enzyme-free dissociation buffer supports pluripotency of the generated iPSCs for more than 50 passages.

引言

Genomic comparisons of human and non-human primates (NHPs) are crucial to understand our evolutionary history and the evolution of human-specific traits¹. Additionally, these comparisons allow for the inference of function by identifying conserved DNA sequences², e.g., to prioritize disease-associated variants³. Comparisons of molecular phenotypes such as gene expression levels are crucial to better interpret genomic comparisons and to discover, for example, cellular phenotypic differences. Furthermore, they have - similar to comparisons at the DNA level - the potential to infer functional relevance, and hence to better interpret medically relevant variation within humans⁴. The incorporation of comprehensive molecular phenotypic data into these comparative studies requires appropriate biological resources (i.e., orthologous cells across species). However, ethical and practical reasons make it difficult or impossible to access such comparable cells, especially during development. Induced pluripotent stem cells (iPSCs) allow for the generation of such inaccessible cell types in vitro^5,6, are experimentally accessible, and have been used for primate comparisons^{6,7,8,9,10,11,12,13,14}.

To generate iPSCs, one needs to acquire the primary cells to be reprogrammed. Cells isolated from urine have the advantage that they can be sampled non-invasively from primates, and that they can be readily reprogrammed, probably due to their stem cell-like molecular profiles¹⁵. The culture conditions to maintain primate iPSCs are as important as reprogramming; classically, the culture of human pluripotent stem cells required a non-defined, serum-based medium and co-culture of mouse embryonic fibroblasts - so-called feeder cells - that provide essential nutrients and a scaffold for embryonic stem cells (ESCs)¹⁶. Since the development of chemically defined and feeder-free culture systems^17,18, there are now various options of commercially available iPSC culture media and matrices. However, most of these culture conditions have been optimized for human ESCs and iPSCs, and hence might work less well in NHP iPSC culture. In this video protocol, we provide instructions to generate and maintain human and NHP iPSCs derived from urinary cell culture.

Since the first report of iPSC generation by the forced expression of defined factors in fibroblasts in 2006, this method has been applied to many different cell types of various origins^{19,20,21,22,23,24,25,26,27,28,29,30,31,32}. Among them, only urine-derived cells can be obtained in a completely non-invasive manner. Based on the previously described protocol by Zhou et al.³³, one can isolate and expand cells from primate urine even from non-sterile samples, by supplementing broad-spectrum antibiotics¹⁵. Notably, urine-derived cells sampled by this protocol exhibit a high potential to produce iPSCs, within a shorter period of time (colonies become visible in 5-15 days) than the conventional reprogramming of fibroblasts (20-30 days, in our experience), and with a sufficiently high success rate. These urine-derived cells were classified as the mixed population of mesenchymal stem cell-like cells and bladder epithelial cells, causing the high reprogramming efficiency¹⁵.

In addition to the variation in primary cells, the reprogramming methods to generate iPSCs also vary according to the purpose of usage. Conventional reprogramming procedures for human somatic cells were carried out by the overexpression of reprogramming factors with retrovirus or lentivirus vectors, which allowed the integration of exogenous DNA in the genome^5,34,35. To keep the generated iPSCs genomically intact, researchers have developed a wide variety of non-integration systems - excisable PiggyBac vector^36,37, episomal vector^38,39, non-integrating virus vectors such as Sendai virus⁴⁰ and adenovirus⁴¹, mRNA transfection⁴², protein transfection^43,44, and chemical compound treatment⁴⁵. Due to the efficiency and ease in handling, the Sendai virus-based reprogramming vectors are used in this protocol. Infection of primary cells is performed in a 1 h suspension culture of cells and viruses at a multiplicity of infection (MOI) of 5 prior to plating. This modified step could increase the likelihood of contact between cell surfaces and viruses, compared to the conventional method in which the viruses are added directly to the adherent cell culture, and thus yield more iPSC colonies¹⁵.

Passaging of human and NHP pluripotent stem cells can be done by clump passaging and single-cell passaging. Ethylenediaminetetraacetic acid (EDTA) is a cost-efficient chelating agent that binds calcium and magnesium ions, and thus prevents the adherent activity of cadherin and integrin. EDTA is also used as a mild, selective dissociation reagent, as undifferentiated cells detach before differentiated cells due to their different adhesion molecules. Complete dissociation induces massive cell death of primate iPSCs via the Rho/Rho-associated coiled-coil containing protein kinase (Rho/Rock)-mediated myosin hyperactivation. Therefore, supplementing the culture medium with a Rho/Rock inhibitor is essential for experiments that require single cells in suspension^46,47. In this protocol, we recommend clump passaging as the routine passaging method and recommend single-cell passaging only when it is necessary, e.g., when seeding of defined cell numbers is required, or during sub-cloning.

方案

This experimental procedure was approved by the responsible ethic committee on human experimentation (20-122, Ethikkommission LMU München). All experiments were performed in accordance with relevant guidelines and regulations.
NOTE: Approval must be obtained from the appropriate ethical committee before starting experiments dealing with human and NHP samples. All experimental procedures must be performed in accordance with relevant guidelines and regulations. Each of the following steps should be performed using sterile technique in a biological safety cabinet. All buffer and media compositions can be found in Supplementary Table S1. Ensure that all media are warmed to room temperature (22 °C) before being added to the cells. Each centrifugation step should be performed at room temperature, unless mentioned otherwise.

1. Isolation of cells from urine samples

CAUTION: Ensure that human donors are free from human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV). For NHPs, make sure the possible donors/cells are free from specific pathogens-B Virus (BV), Simian Immunodeficiency Virus (SIV), Simian Betaretrovirus (SRV), and Simian T Cell Lymphotropic Virus (STLV).

1. Prepare a gelatin-coated 12-well plate by adding 500 µL of 0.2% gelatin per well, and distribute the liquid by moving the plate. Place at 37 °C for at least 30 min before needed.
2. Collect human urine samples in 50 mL conical tubes. For primates, collect urine from the floor of the animal facility with a syringe.
   NOTE: A volume of 5 mL of urine was proven to be sufficient for isolating at least one colony in 42% of the attempts. However, using a higher volume of ~50 mL of urine is recommended to increase the chance of isolating colonies. NHP urine should be sampled as fresh as possible, preferably immediately after urination. The storage of urine samples at 4 °C for 4 h had no negative effect on the success rate of the protocol, but longer storage times were not tested.
3. Centrifuge the urine-containing tube at 400 × g for 10 min, and carefully aspirate the supernatant, leaving approximately 1 mL in the tube.
4. Resuspend the pellet in the residual 1 mL of liquid. Pool the suspensions in one tube if multiple tubes of urine were collected.
5. Wash the cells by adding 10 mL of urine wash buffer (see Supplementary Table S1) containing 2.5 µg/mL amphotericin to the tube, and carefully mix the suspension using a serological pipette.
6. Centrifuge the tube at 200 × g for 10 min, and carefully aspirate the supernatant, leaving approximately <0.2 mL in the tube.
7. Resuspend the cell pellet in 1 mL of primary urine medium (see Supplementary Table S1) containing 0.5 µg/mL amphotericin per 50 mL of initially processed urine (resuspend in 1 mL, even if less than 50 mL of urine was processed).
8. Aspirate gelatin from the wells (prepared in step 1.1), and plate 1 mL of the suspension from step 1.7 into one well of a 12-well plate. Repeat for as many wells as desired, or for as many mililiters of suspension available.
   Optional: To avoid contamination originating from unsanitary sample collection, add 100 µg/mL antimicrobial reagent to the cells from here on, until the first passage.
9. Place the plate in a 37 °C, 5% CO₂ incubator.
10. Add 1 mL of primary urine medium per well daily until day 5, without removing the existing medium.
11. On day 5, aspirate 4 mL of medium from the plate, leaving approximately 1 mL of medium. Add 1 mL of REMC medium (see Supplementary Table S1) per well to get a 1:1 mixture with the new culture medium.
12. Replace half of the medium with REMC medium every day until the first colonies appear (Figure 1A, B). Therefore, remove 1 mL of old medium, and add 1 mL of fresh REMC medium per well.

2. Expansion of urinary cells

NOTE: Urinary cell passaging should be conducted before the culture reaches 90% confluency.

1. Prepare the desired amount of gelatin-coated 12-well plates, as stated in step 1.1.
2. Aspirate the old medium, and wash the cells by adding 1 mL of Dulbecco's phosphate buffered saline (DPBS).
3. Aspirate the DPBS, and add 300 µL of 0.5x dissociation enzyme diluted with DPBS. Incubate the plate at 37 °C for 5 min.
4. Add 700 µL of REMC medium to stop the enzymatic reaction. Gently pipette the suspension using a P1000 pipette until the cells are dissociated into single cells.
5. Transfer the cell suspension to a 15 mL tube, and centrifuge the tube at 200 × g for 5 min.
6. Carefully aspirate the supernatant and resuspend the pellet in 1 mL of REMC medium.
7. Count the cells using a cell counter (a hemocytometer or an automated cell counter).
8. For expansion of the urinary cells, plate 1.5 × 10⁴ to 3 × 10⁴ cells in 1 mL of REMC medium into one 12-well plate coated with 0.2% gelatin.
9. Perform subsequent medium changes every other day until the culture reaches 80%-90% confluency. Therefore, aspirate the old medium and add 1 mL of fresh REMC medium.

3. Generation of iPSCs by Sendai virus vector infection

NOTE: For the workflow of the reprogramming procedure, see Figure 2A. Urinary cells used for reprogramming should be as young as possible, but a remarkable loss of reprogramming efficiency is not observed before passage 4. The Sendai Virus Reprogramming Kit must be used in a BL-2 facility. Handle viruses under a biological safety cabinet with laminar flow, and always use appropriate safety equipment to prevent mucosal exposure.

1. Prepare a basement membrane matrix-coated 12-well plate by adding 500 µL of basement membrane matrix per well, and distribute the liquid by moving the plate. Incubate the plate at 37 °C for at least 1 h, and replace the basement membrane matrix with 900 µL of REMC medium. Store the plate at 37 °C until use.
2. Quickly thaw the components of the Sendai Reprogramming Kit in a 37 °C water bath. Mix the Sendai viruses (polycistronic KLF4-OCT3/4-SOX2, cMYC, and KLF4) with a MOI of 5, and add REMC medium up to 100 µL. Use equation (1):
   
   NOTE: As virus titers differ between lots, always check the titer in the certificate of analysis that is provided by the manufacturer.
   Optional: Use green fluorescent protein (GFP) Sendai virus in addition as a positive control for the transduction efficiency. For this, prepare an additional 3.5 × 10⁴ cells in a separate tube during step 3.3.
3. For dissociation of the urinary cells, follow steps 2.2-2.4. Count the cells using the cell counter, and transfer 7 × 10⁴ urinary cells to a 1.5 mL tube.
4. Centrifuge the tube at 200 × g for 5 min, and carefully remove the supernatant without disrupting the cell pellet. Resuspend the pellet in 100 µL of the SeV mixture prepared in step 3.2. Incubate the tube for 1 h at 37 °C for suspension infection.
5. Plate the suspension on the basement membrane matrix-coated 12-well plates that were prepared in step 3.1. Routinely, plate 1 × 10⁴ and 2.5 × 10⁴ cells per well in duplicates.
6. Incubate the cells at 37 °C and 5% CO₂. Replace the medium with 1 mL of fresh REMC medium 24 h post-transduction and on day 3.
7. On day 5 after transduction, change the medium to PSC generation medium (see Supplementary Table S1), with subsequent medium changes every other day. Therefore, remove the old medium and add 1 mL of PSC generation medium per well.
   NOTE: It can take up to 15 days until the first colonies appear.
8. Pick individual iPSC colonies when the size of the colony exceeds 1 mm. To do this, scrape and carefully collect a single colony with a p10 pipette under a microscope. Transfer the colony into a new well of a 12-well plate coated with a basement membrane matrix containing 750 µL of PSC culture medium.
   Optional: Rinsing the plate with DPBS and treating for 1 min with 0.5 mM EDTA prior to picking could support the robust culture of further steps. If the cells are to be cultured for longer to wait for later emerging colonies, do not perform this EDTA treatment step.
9. Grow the cells at 37 °C and 5% CO₂ with subsequent medium changes every other day, as stated in section 4 of the protocol. When the picked colony reaches a diameter of 2 mm, continue with the routine iPSC passaging, as explained in section 5 of the protocol.

4. Medium change

NOTE: The culture medium should be changed every other day until the colonies grow large enough for passaging.

1. Aspirate the old medium and add 750 µL of the fresh medium per 12-well plate. To switch to a different type of medium, replace the medium at least 1 day after passaging.

5. Passaging

NOTE: The cells should be passaged when the iPSC colonies grow large enough (diameter > 2 mm), or the colonies are about to touch each other. Routinely, iPSCs can be split approximately every 5 days. Use clump-passaging (step 5.1) for routine maintenance, and single-cell passaging (step 5.2) for experiments where a defined number of cells is needed. In case the iPSCs differentiate a lot, colony picking (step 5.3) can help improve the purity of the cultures.

1. Clump-passaging
   1. Prepare a basement membrane matrix-coated 12-well plate by adding 500 µL of basement membrane matrix per well, and distribute the liquid by moving the plate. Incubate the plate at 37 °C for at least 1 h. Replace the basement membrane matrix with 500 µL of PSC culture medium and store the plate at 37 °C until use.
   2. Aspirate the medium from the cultured cells, and wash the cells by carefully adding 500 µL of DPBS. Remove the DPBS and add 500 µL of 0.5 mM EDTA to the well.
   3. Incubate the plate at RT for 2-5 min, until the colonies start detaching. Carefully observe the cells under the microscope.
   4. When the edges of the colonies start to peel off and gaps between the cells become visible (Figure 3A), remove the EDTA and carefully add 500 µL of DPBS.
      NOTE: Always pipette against the side wall of the well and never directly onto the cells, so as not to detach the cells from the plate.
   5. Aspirate the DPBS and flush the well with 500 µL of PSC culture medium using a p1000 pipette. Pipette up and down 1x-5x to disperse the colonies into clumps of appropriate size (Figure 3A). Do not pipette too much.
      ​NOTE: If the iPSCs are accidentally pipetted too much, add 10 µM of Rock inhibitor Y-27632 to the medium. This can enhance survival, as iPSCs are not able to survive as single cells.
   6. Transfer 1/10-1/50 of the cell clump suspension to the new wells. The ratio depends on the confluency of the well before splitting, the desired density of the seeded cells, and iPSC clonal preference.
   7. Distribute the clumps evenly in the well by gently moving the plate back and forth several times. Incubate the plate for at least 30 min at 37 °C to let the clumps attach.
   8. Replace the medium with 750 µL of PSC culture medium if many floating dead cells are observed; otherwise, add 250 µL of PSC culture medium. Place the plate at 37 °C and 5% CO₂ in an incubator.
      ​NOTE: Medium replacement after 30 min is critical, especially for unstable cell lines (e.g., NHPs).
   9. Change the medium every 2-3 days until the colonies grow large enough for passaging. For medium change, follow step 4 of the protocol.
2. Single-cell passaging
   1. Prepare the basement membrane matrix-coated culture plate, as stated in step 5.1.1, with the addition of 10 µM Y-27632 to the PSC culture medium.
      Optional: Add 10 µM Y-27632 to the cells 1-3 h prior to passaging to enhance the survival of sensitive cell lines.
   2. Aspirate the medium and wash the cells by adding 500 µL of DPBS. Remove the DPBS and add 300 µL of detachment solution to the wells.
   3. Incubate the plate at 37 °C for 5-10 min. When sufficient detachment of the cells is observed under the microscope, add 700 µL of PSC culture medium or DPBS.
   4. Pipette up and down 5-10x using a p1000 pipette until the cells are dissociated into single cells. Do not pipette too much, in order to prevent cell damage.
   5. Transfer the cell suspension to a 15 mL tube containing at least 2 mL of DPBS to dilute the detachment solution.
   6. Centrifuge the tube at 200 × g for 5 min and aspirate the solution completely, without disrupting the cell pellet.
   7. Resuspend the pellet in 500 µL of PSC culture medium supplemented with 10 µM Y-27632.
   8. Count the cells and seed 5,000-7,000 cells per basement membrane matrix-coated 12-well plate, prepared in step 5.2.1.
      NOTE: If a different cell number is needed, change to a bigger or smaller well accordingly.
   9. Incubate the plate for at least 30 min at 37 °C and 5% CO₂ to let the cells attach.
   10. Replace the medium with 750 µL of PSC culture medium + 10 µM Y-27632 if many dead cells are observed; otherwise, add 250 µL + 10 µM Y-27632.
       ​NOTE: This step is critical, especially for the unstable cell lines (e.g., NHPs).
   11. Place the plate at 37 °C and 5% CO₂ in an incubator.
   12. Change the medium to PSC culture medium without Y-27632 1 to 2 days after splitting, to allow the cells to display the classic colony morphology again (Figure 3B).
   13. Change the medium every 2 days until the colonies grow large enough. For medium change, follow section 4 of the protocol.
3. Passaging of iPSCs by colony picking
   1. Prepare basement membrane matrix-coated 12-wells as stated in step 5.1.1.
   2. Aspirate the medium and wash the cells by carefully adding 500 µL of DPBS. Remove the DPBS and add 500 µL of 0.5 mM EDTA to the well.
   3. Incubate the plate at RT for 1-3 min and observe the cells under the microscope, until detachment of the colony is visible on the borders.
   4. Remove the EDTA and carefully add 500 µL of DPBS. Aspirate the liquid before slowly adding 500 µL of PSC culture medium to the well, without detaching the cells.
   5. Use a p200 pipette to pick the desired colony under the microscope, without collecting the differentiated cells. To do this, gently scratch over the colony while taking up the medium containing cells.
   6. Transfer each picked colony into one basement membrane matrix-coated well, as prepared in step 5.3.1. Dissociate the cells into small clumps using a p1000 pipette, by pipetting the cells 2-5x.
   7. Incubate the plate for 30 min at 37 °C and 5% CO₂, allowing the clumps to attach.
   8. Replace the medium with 750 µL of PSC culture medium if many floating dead cells are observed; otherwise, add 250 µL of PSC culture medium.
      ​NOTE: Medium replacement after 30 min is critical, especially for the unstable cell lines (e.g., NHPs).
   9. Place the plate at 37 °C and 5% CO₂ in an incubator.
   10. Change the medium every 2-3 days until the colonies grow large enough for passaging. To do this, follow section 4 of the protocol.

6. Freezing of urinary cells and iPSCs for long-term storage

NOTE: Routinely, iPSCs are frozen as clumps in cell freezing medium without counting. Pipetting should be minimal, to avoid dissociation into single cells. For urinary cells, routinely, 1.5 × 10⁴ to 3 × 10⁴ cells are frozen per tube, allowing the user to thaw one tube directly in one well of a 12-well plate without the need of another counting step.

1. Prepare 5 mL of DPBS in a 15 mL tube.
2. For the freezing of urinary cells, follow steps 2.2-2.4 of the protocol. For the freezing of iPSCs, follow steps 5.1.2-5.1.5 from the clump passaging protocol.
3. Transfer the suspension to the 15 mL tube prepared in step 6.1. For the freezing of urinary cells, count 10 µL of the cell suspension using a hemocytometer. Centrifuge the cells for 5 min at 200 × g, and aspirate the supernatant completely.
4. Resuspend the cell pellet in 400 µL of cell freezing medium per tube, and distribute the cells to the desired amount of cryotubes.
5. Transfer the cryotubes immediately to -80 °C. Transfer the frozen tubes to a -150 °C freezer or liquid nitrogen 1 day after freezing at -80 °C for long-term storage.

7. Thawing of urinary cells and iPSCs

1. For the thawing of urinary cells, prepare the desired amount of gelatin-coated 12-wells, as stated in step 1.1 of the protocol. For iPSCs, prepare the basement membrane matrix-coated 12-well plates, as stated in step 5.1.1. In both cases, do not exchange the matrix with medium.
2. Prepare a 15 mL tube containing 4 mL of DPBS, and store it at 37 °C.
3. Place a frozen vial of cells quickly in a 37 °C water bath for thawing, until a piece of floating ice becomes visible.
   NOTE: Wipe the cryotube with ethanol before and after incubation in the water bath to avoid contaminations.
4. Add 500 µL of REMC medium for urinary cells, or 500 µL of PSC culture medium for iPSCs to the ice-containing suspension, and transfer the suspension immediately to the pre-warmed 15 mL tube prepared in step 7.2.
5. Centrifuge the tube at 200 × g for 5 min, and discard the supernatant completely.
6. For urinary cells, resuspend the pellet in 1 mL of REMC medium. For iPSCs, carefully resuspend the pellet in 750 µL of PSC culture medium. Avoid pipetting too much, in order to keep the clumps intact.
   Optional: Supplementing the medium with 10 µM Y-27632 can support the survival of iPSCs after thawing.
7. Aspirate the matrix from the 12-well plates prepared in step 7.1, and carefully transfer the cell suspension to the well.
8. Place the plate overnight at 37 °C and 5% CO₂ in an incubator.
9. The next day, replace the medium with PSC culture medium, without Y-27632 for iPSCs and with REMC for urinary cells.
10. Grow the cells at 37 °C and 5% CO₂ in an incubator.
11. Change the medium every 2-3 days until the cells grow large enough for passaging. For medium change, follow section 4 of the protocol.

8. Immunocytochemistry

NOTE: Immunostaining with antibodies targeting pluripotency-related markers such as NANOG, OCT3/4, SOX2, TRA-1-60 and EpCAM is one of the most widely used validations of newly generated iPSCs. Further information about the antibodies and dilutions can be found in the Table of Materials.

1. Plate iPSCs 1-3 days prior to use in an appropriate number of 12-well plates. Aspirate the medium, wash the cells by adding 500 µL of DPBS, and remove the DPBS. Add 400 µL of 4% paraformaldehyde (PFA) per well, and fix the cells for 15 min at RT.
2. Remove the 4% PFA, and wash the cells 3x with DPBS. Add 400 µL of blocking buffer per well, and incubate the plate for 30 min at RT.
3. Aspirate the blocking buffer, and add the antibodies diluted in 400 µL of antibody dilution buffer (ADB) to each well. Incubate the plate at 4 °C overnight.
4. Remove the ADB containing the primary antibodies, and wash cells 3x with DPBS.
5. Aspirate the DPBS, and add 400 µL of secondary antibodies diluted in ADB per well. Incubate the plate for 1 h at RT in the dark.
6. Remove the ADB, and wash cells 3x with DPBS. Add 1 µg/mL 4',6-diamidino-2-phenylindole (DAPI) diluted in DPBS per well, and incubate for 3 min at RT.
7. Aspirate the DAPI solution, and wash the cell 3x with DPBS. Add 500 µL of DPBS for imaging.

结果

When isolating cells from human and NHP urine, different types of cells can be identified directly after isolation. Squamous cells, as well as various smaller round cells, get excreted with the urine; female urine contains far more squamous cells than male urine (Figure 1B - Day 0; Supplementary Figure S1). After 5 days of culture in primary urine medium, the first adherent proliferating cells can be seen (Figure 1A,B - Day 5). ...

讨论

iPSCs are valuable cell types as they allow the generation of otherwise inaccessible cell types in vitro. As the starting materials for reprogramming, for example, fibroblasts are not easily available from all primate species, this paper presents a protocol for the generation of iPSCs from urine-derived cells. These cells can be obtained in a non-invasive manner, even from non-sterile primate urine samples, by supplementing the culture medium with broad-spectrum antibiotics.

Several c...

材料

本文使用的材料清单

姓名	公司	目录编号	评论
Accumax™ 细胞分离溶液 （Detachment solution）	Sigma-Aldrich	SCR006
两性霉素 B 溶液	默克	A2941-100ML
抗人 TRA-1-60 小鼠抗体	Stem Cell Technologies	60064	稀释度：1/200
抗人 TRA-1-60 PE 偶联抗体	Miltenyi Biotec	130-122-965	稀释度：1/50
Bambanker™（细胞冷冻培养基）	Nippon Genetics	BB01
牛血清白蛋白 （BSA）	Sigma-Aldrich	A3059-100G
细胞培养多孔板，12 孔 CELLSTAR	Greiner BIO-ONE	665180
伯爵夫人™II 自动细胞计数仪	Thermo Fisher Scientific	AMQAX1000
CryoKing® 1.5 mL 试管（带 2D 条形码冻存管）	Sued-Laborbedarf	52 95-0213	不同类型的冻存管可用于冻存。2D 条形码试管的优点是样品信息可以存储在具有唯一试管信息的数据库中。
CytoTune™EmGFP 仙台病毒报告基因 （GFP 仙台病毒）	Thermo Fisher Scientific	A16519
CytoTune™-iPS 2.0 仙台病毒重编程试剂盒（ 仙台病毒重编程试剂盒）	Thermo Fisher Scientific	A16518
DAPI 4'，6-二脒-2'-苯基吲哚二盐酸盐	Sigma-Aldrich	10236276001
DMEM High葡萄糖	TH。Geyer	L0102
DMEM/F12 w L-谷氨酰胺	Fisher Scientific	15373541
驴抗小鼠 IgG （H+L） 高度交叉吸附二抗，Alexa Fluor™ 488	Thermo Fisher Scientific	A-21202	稀释度：1/500
驴抗兔 IgG （H+L） 高度交叉吸附二抗，Alexa Fluor™ 594	Thermo Fisher Scientific	A-21207	稀释度：1/500
DPBS 不含钙，不含镁	TH。Geyer	L0615-500
EpCAM 重组多克隆兔抗体 （22 HCLC）	Thermo Fisher Scientific	710524	稀释度：1/500
乙二胺四乙酸 （EDTA）	Carl Roth	CN06.3
Falcon 管 15 mL 锥形底	Bio-ONE	188271-N
Falcon 管 50 mL 锥形底	Bio-ONE	227261
胎牛血清，合格，热灭活，巴西 （FBS）	Thermo Fisher Scientific	10500064
FlowJo V10.8.2	FlowJo
猪皮明胶	Sigma-Aldrich	G1890-1KG
Geltrex™无 LDEV、经 hESC 验证、低生长因子基底膜基质	Thermo Fisher Scientific	A1413301
GlutaMAX™补充	Thermo Fisher Scientific	35050038
Heracell™ 240i CO2培养箱	Fisher Scientific	16416639
Heraeus HeraSafe安全柜	Kendro	51017905
人EGF，优质级	Miltenyi Biotec	130-097-749
ImageJ	斐济	2.9.0
MEM 非必需氨基酸溶液 （100X）	Thermo Fisher Scientific	11140035
微量离心管 PP，1.5 mL	Nerbe Plus	04-212-1000
显微镜 Nikon eclipse TE2000-S	Nikon	TE2000-S
小鼠抗 α-胎蛋白抗体	R&D Systems	MAB1368	稀释度：1/100
小鼠抗 α-平滑肌肌动蛋白抗体	R&D Systems	MAB1420	稀释度：1/100
小鼠抗 β-III 微管蛋白抗体	R&D Systems	MAB1195	度：1/100
mTeSR™1	STEMCELL Technolgies	85850
Nanog （D73G4） XP 兔单克隆抗体	Cell Signaling Technology	4903S	稀释度：1/400
Normocure™（抗菌试剂）	Invivogen	ant-noc
Oct-4 兔抗体	Cell Signaling Technology	2750S	稀释度：1/400
多聚甲醛 （PFA）	Sigma-Aldrich	441244-1KG
青霉素-链霉素 （10.000 U/ml） （PS）	Thermo Fisher Scientific	15140122	青霉素-链霉素混合物含有 100 U/mL 青霉素和 100 &微量;g/mL 链霉素。
重组人 FGF-basic	PeproTech	100-18B
重组人 PDGF-AB	PeproTech	100-00AB
冷冻台式离心机	SIGMA	4-16KS
肾上皮细胞基础培养基	ATCC	PCS-400-030
肾上皮细胞生长试剂盒	ATCC	PCS-400-040
Sox2 （L1D6A2） 小鼠单克隆抗体 #4900	Cell Signaling Technology	4900S	稀释度：1/400
SSEA4 （MC813） 小鼠单克隆抗体	NEB	4755S	稀释度：1/500
StemFit®Basic02	Nippon Genetics	3821.00	该培养基已停止生产，使用StemFit Basic04CT检测人类细胞系或StemFit Basic03检测非人灵长类动物。
Triton X-100	Sigma-Aldrich	T8787-50ML
TrypLE™选择酶 （1x），无酚红（解离酶）	Thermo Fisher Scientific	12563011
水浴精密 GP 05	Thermo Fisher Scientific	TSGP05
Y-27632，二盐酸盐（Rock 抑制剂）	Biozol	BYT-ORB153635
抗体稀释缓冲液			有关成分，请参见补充表 S1
封闭缓冲液			成分见附表 S1
REMC 培养基			成分见附表 S1
原代尿培养基			成分见附表 S1
PSC 培养基			见附表 S1
PSC 生成培养基			成分见附表 S1
洗涤液			成分见附表 S1