Introducing the Stem Cell ASCL2 Reporter STAR into Intestinal Organoids

Summary Patient-derived organoids maintain functional and phenotypic characteristics of the original tissue such as cell-type diversity. Here, we provide protocols on how to label intestinal (cancer) stem cells by integrating the stem cell ASCL2 reporter (STAR) into human and mouse genomes via two different strategies: (1) lentiviral transduction or (2) transposon-based integration. Organoid technology, in combination with the user-friendly nature of STAR, will facilitate basic research in human and mouse adult stem cell biology. For complete details on the use and execution of this protocol, please refer to Oost et al. (2018).

c. STAR can also be used to mark stem-like cells in colorectal cancer. Recent evidence suggests that not all patient-derived colorectal cancer cell lines express ASCL2 (Jubb et al., 2006;Ziskin et al., 2013), which constitutes a natural but hitherto not encountered limit. d. STAR might be suitable to reflect the transcriptional activity of ASCL2 in organoid systems beyond the intestine of men and mice. However, this has not been tested yet. e. When STAR is expected to target a non-stem cell population, a ubiquitously expressed selection cassette is preferable. For more details see section ''Selection Procedure for STAR Organoids.'' f. As Wnt signaling drives ASCL2 expression (Van der Flier et al., 2007), supplementing the organoid medium with exogenous Wnt may lead to a high fraction of STAR + cells with moderate differences in STAR levels. Cellular differentiation protocols can lead to a higher degree of cellular diversity by reducing the amount of stemness-promoting factors. Consequently, these media induce crypt-(like) budding structures and promote the difference between STAR-high and STAR-negative cells (Sato et al., 2011). g. Studies previously performed using STAR organoids: STAR has been used to study the cellular composition of human colon and patient-derived CRC organoids. Colony formation assays have demonstrated the stem cell properties of single STAR + cells in human colon organoids. Additionally, STAR has been used to profile tumor subpopulations transcriptomically and to visualize cancer stem cells in xenograft studies (Oost et al., 2018). 2. Comparison of STAR to endogenous labeling of stem cells: a. STAR is a very versatile tool, allowing to generate a large number of reporter lines simultaneously in a relatively short time frame with a high success rate (thus maintaining polyclonality). b. As STAR is reporting the transcriptional activity of ASCL2, the brightness of the fluorescent protein is not limited by minimum expression level of an endogenous stem cell marker gene. This makes STAR superior for time-lapse microscopy studies and studies on cell fate conversions (differentiation or plasticity). c. Visualization of intestinal stem cells in human organoids has been performed, for instance by generating a CRISPR/Cas9-mediated knockin into the LGR5 locus (Fujii et al., 2018). By endogenously tagging the LGR5 protein with a fluorescent protein, both the subcellular localization and interaction partners of LGR5 can be reliably studied. d. Genetic lineage tracing studies provide easy readout for assessing a cell's self-renewal and multipotency potential. Using LoxP-STOP-LoxP-based reporter systems and a knockin of CreER into the LGR5 locus, this has been successfully performed in human colonic organoids Sugimoto et al., 2017). 3. Cellular localization of the fluorescent protein: We have generated STAR plasmids with and without a nuclear localization signal (NLS) fused to the fluorescent protein.
Their key features are listed below: a. Nuclear localization i. Excellent for quantifications and cell tracking ii. In combination with a bright fluorophore, more difficult to distinguish expressions levels with basic microscopy as the fluorescent proteins are concentrated in a smaller volume b. Uniform localization i. Displays cellular morphology (e.g., stem cells are slender cells in mouse small intestinal organoids) ii. Excellent for co-expression studies with cytoplasmic (stem cell) markers 4. Features of fluorescent proteins a. To capture ASCL2 dynamics with high accuracy during time-lapse microscopy, the ideal fluorophore would have to have a high extinction coefficient and quantum yield (brightness), high List of all available STAR plasmids generated by the Snippert lab and provided to Addgene. Plasmids are either suitable for lentiviral-based (pLV) or transposon-based (Tol2) integration. The fluorescent color reporting STAR activity and selection antibiotics are listed for each plasmid. For a graphical representation of the STAR plasmids see Figure 2.

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photostability (limited bleaching), a short maturation time, and a high protein turnover. Most of these criteria are met by either mNeonGreen or sTomato, however, depending on the research question a different fluorophore might be more suitable. b. When planning to use the STAR lines for in vivo experiments, using a fluorescent protein with an established antibody provides highest flexibility in terms of downstream processing and allows, for instance, for paraffin-embedding or Edu chemistry and subsequent retrieval of the STAR signal. 5. Microscope setup Generally speaking, the different fluorophores used in this study are suitable for many microscopy techniques as the proteins can be excited with either of the following laser options: 405 nm, 458 nm, 488 nm, or 561 nm. If, however, STAR shall be combined with multiple different colors which leads to limited detection windows, it might be preferable to choose bright fluorescent proteins which can be most optimally excited with the available laser equipment.

Choosing Your Integration Strategy
We provide STAR plasmids that can be integrated into the genome of organoids either by lentiviral transduction or by transposase-based approaches. All three integration strategies are suitable for the generation of organoid lines with the following features: species: mouse or human, organ: small intestine or colon, genome: WT or cancerous. In our experience, organoid lines with a WT genome might be more difficult to transfect with a transfection reagent (approach C). Hence, in situations transposon-based integration with electroporation might be superior (approach B). An overview of the experimental workflows is provided in Figure 1.

Preparation of Organoid Cultures
Timing: 1-2 weeks To maximize the efficiency of generating stable STAR organoids, the organoids should be in a healthy, proliferative state prior to starting any transduction/transfection protocol ( Figure 3).
CRITICAL: (for mouse SI organoids) We recommend reverting mouse SI organoids from their regular crypt-like morphology (ENR culture medium) into a highly proliferative state (WENR culture). To get to this state, mouse SI organoids should be cultured in WENR medium (see section ''Materials and Equipment / General Organoid Media'') after passaging by mechanically shearing off the crypt units. When most of the organoids demonstrates a cystic morphology (usually after 5-10 days), organoids can be used for STAR integration ( Figure 3C).
For complete details on the use and execution of this protocol paragraph, please refer to Koo  Note: To obtain enough starting material, it is advisable to expand the organoid culture for 1-2 weeks prior to STAR integration. For electroporation-based transfection, the success rate increases significantly if 500,000-1 million cells are being electroporated. This corresponds to organoids growing in approx. 200-400 mL Matrigel for 7-10 days after the last passage.

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CRITICAL: (for transfection-based approaches) As serum hampers the transfection efficiency, organoids should be cultured with serum-free medium for the transfection-based approaches (procedures B and C), starting one day in advance. This implies leaving out Penicillin/Streptomycin and replacing the Wnt CM with 10 mM CHIR99021 (see Step-By- Step Method Details).

MATERIALS AND EQUIPMENT
The STAR Selection A selection of STAR plasmids for either lentiviral or Tol2 transposon-based integration has been generated and is depicted in Figure 2 and Table 1. The plasmid sequences at base-pair resolution are available on Addgene.

STEP-BY-STEP METHOD DETAILS
Integration of STAR into the Genome The protocols below describe the integration of STAR via (A) lentiviral transduction, (B) transposase-based integration with electroporation, and (C) transposase-based integration using a transfection reagent.

Protocol A: Lentiviral Transduction
Timing: 5 days

Protocol A1: Production of Lentiviral Particles
This protocol describes the production of lentiviral particles using VSV-G envelopes. All lentiviral STAR plasmids listed in Table 1 are suitable for 3rd generation lentivirus making with VSV-G envelopes and the packaging vectors gag, pol, and rev. Transduced stem cells will generate STAR organoids.
CRITICAL: Protocol steps involving lentiviral particles must be performed in a BSL-2 lab (indicated below). This involves careful attention to BSL-2 regulations on sample handling and disposal of plasticware and media.
Note: Each plate at 80% confluency can be split over 4 plates. 4. Drip 127 mL DNA transfection solution onto HEK293T cells growing in DMEM/FBS and store them in a humidified cell culture incubator at 37 C.
Day 3: Timing: 5 min CRITICAL: The following step must be performed in a BSL-2 lab.
5. Carefully aspirate medium from HEK293T culture plates and add 8 mL DMEM/FBS to each plate.
Note: This culture media volume allows to pool the virus suspension of up to 4 plates into one bucket for ultracentrifugation on Day 5.

Day 5:
Timing: 3.5 h, including 2.5 h centrifugation CRITICAL: The following steps must be performed in a BSL-2 lab.
6. Before you begin: a. Pre-cool ultracentrifuge to 4 C b. Prepare transduction medium as described in section ''Additional Organoid Media Required for Lentiviral Transduction (Protocol A)'' c. Optional: Prepare freezing medium for lentiviruses as described in section ''Additional Organoid Media Required for Lentiviral Transduction (Protocol A)'' 7. Optional: If dead cells are floating in the culture medium, collect supernatant in 15 mL flask and centrifuge for 5 min at 500 3 g. Continue with supernatant. 8. Collect the culture medium from the plates and push it through a 0.45 mm filter using a large 60 mL syringe. 9. Load ultracentrifuge buckets with tubes and pipet virus suspension into the tube.
Note: Culture media of up to 4 plates can be combined into one ultracentrifugation bucket.

Carefully equal balance opposite buckets (lids included) by topping up with DMEM/FBS.
Afterwards, carefully cap buckets with corresponding lids. 11. Centrifuge for 2.5 h at 170,000 3 g at 4 C. 12. In the meantime, prepare N*550 mL transduction medium with N being the total number of flasks or plates in which virus has been produced. 13. Transfer ultracentrifuge buckets very carefully into a laminar flow hood, remembering the orientation of the tube inside the centrifuge. 14. Open bucket containing ultracentrifuge tube and decant medium carefully in such orientation that the opaque brown pellet is on the upper side of the tube. Take a micropipette and remove leftover medium, while taking care not to agitate the pellet. 15. Resuspend the virus pellet in N*500 mL of organoid transduction medium. 16. Use 250 mL virus suspension (N/2) to transduce organoids grown in 50-100 mL Matrigel (see protocol A2). Residual virus suspension can be aliquoted in single-use fractions (250 mL each) and frozen at À80 C.

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STAR Protocols 1, 100126, December 18, 2020 Pause Point: The lentivirus can be frozen at À80 C and transduction can be done on a different day with a minor loss in efficiency.
Note: In our experience, 2-year old virus stocks are suitable for high efficiency transductions (minor loss compared to fresh virus), when previously stored as single-use aliquots at À80 C.

Protocol A2: Lentiviral Transduction of Organoids
Timing: 3.5-6.5 h, including 1 h centrifugation and 1-4 h incubation CRITICAL: Protocol steps involving lentiviral particles must be performed in a BSL-2 lab (indicated below). This involves careful attention to BSL-2 regulations on sample handling and disposal of plasticware and media.

Before you begin:
a. Pre-heat water bath to 37 C b. Pre-cool centrifuge to 4 C 18. Harvest organoids with ice-cold medium and transfer into a 15 mL tube.
19. Top up to 10 mL with ice-cold Advanced DMEM and spin at 4 C, 300 3 g for 5 min.
Note: Dispase stocks (1003 and 13) can be stored at 4 C but dispase is most active during the first days after preparation.

Prepare transfection medium as described in section ''Additional Organoid Media Required for Transposon-Based Integration (Protocols B and C)''
Protocol B1: Preparation of Organoids for Transfection Day 1: Timing: 5 min 40. Replace organoid medium with transfection medium while using 500 mL transfection medium for each well of a 24-well plate.
Note: 200-400 mL Matrigel containing organoids grown for 7-10 days after the last passage should suffice for one electroporation. Note: Dissociation to small fragments, rather than to single cells, will increase the survival and outgrowth potential upon electroporation. Note: A significant fraction of cells will not survive this harsh procedure. Hence, cells can be plated more densely than usual for maintenance of the organoid line.

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63. Incubate the plate upside-down in the incubator for 10 min until the Matrigel has solidified. 64. Add 500 mL transfection medium + 1.25% DMSO per well for at least 30-60 min. 65. Refresh each well with 500 mL transfection medium without DMSO.

Protocol C: Transposon-Based Integration Using a Transfection Reagent
The protocol for organoid transfection with transfection reagents was inspired by a transfection protocol using liposomes of Schwank et al. (Schwank et al., 2013), while the key transfection step was adapted by the Snippert lab.
67. Pre-heat water bath to 37 C 68. Pre-cool centrifuge to 4 C 69. Prepare 1003 dispase: Dilute 0.05 g Dispase II in 500 mL Advanced DMEM/F12 and mix well by pipetting and vortexing until completely dissolved. 70. Prepare 13 dispase (inside a flow cabinet): Add 100 mL of 1003 dispase stock to 9.9 mL Advanced DMEM/F12 and mix by vortexing briefly.
Note: Dispase stocks (1003 and 13) can be stored at 4 C but dispase is most activeduring the first days after preparation.

Protocol C1: Preparation of Organoids for Transfection
Timing: 3 days Note: Protocol is written for the transfection of one well of a 24-well plate containing 50 mL Matrigel. Sufficient organoid amounts for successful transfection are between 4 and 8 wells of a 24-wells (approx. 200-400 mL Matrigel). 78. Inactivate trypsin by adding 250 mL of trypsin inhibitor (1:1 ratio with trypsin), top up to 10 mL with Advanced DMEM, and spin at 4 C, 500 3 g for 5 min. 79. Take off supernatant and put cells on ice.
81. Mix transfection mixture well by vortexing, spin the tube briefly to remove drops from the lid or the side of the tube, then incubate for 15 min at RT.
CRITICAL: Do not incubate the transfection mixture for more than 30 min.
82. Resuspend cell pellet in 225 mL transfection medium (previously prepared as described at the beginning of this protocol). 83. Add N*53 mL transfection mixture to the cell suspension with N being the total number of wells the organoids were derived from and mix well by pipetting up and down. 84. Incubate for 5-6 h in the incubator with a loosened lid. 85. Toward the end of the incubation period, thaw Matrigel on ice and keep on ice after thawing. 86. Spin tubes at 4 C, 500 3 g for 5 min.

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87. Take off supernatant. 88. Resuspend pellet in Matrigel and plate cells in 10 mL droplets onto a pre-warmed 24-well plate with 4-5 droplets per well. 89. Incubate the plate upside-down in the incubator for 10 min until the Matrigel has solidified. 90. Add 500 mL transfection medium to each well.
Start with selection procedure, 3 days after transfection (see section ''Selection Procedure for STAR Organoids'').

Selection Procedure for STAR Organoids
Timing: 3 days to 2 weeks Selecting for STAR organoids can be started as early as 3 days post integration. If viability is low (usually after electroporation), start selecting 7 days post integration to not further disturb the outgrowth process. Representative figures of organoids prior, during, and after selection are depicted in Figure 4. (The fraction of surviving organoids is representative for STAR integration by transfection. Lentiviral transduction rates usually are between 20%-80% in our hands.) Timing: 5 min 92. Refresh medium and supplement with the appropriate selection antibiotic (puromycin at 1-4 mg/mL and blasticidin at 2-50 mg/mL).
Optional: As the sensitivity to the drugs might vary across lines, a dose-response test on nontransduced/transfected organoids might be informative.
Note: We usually use concentrations which kill off negative clones within 3 and 7 days for puromycin and blasticidin, respectively. In our experience, organoid lines with a WT genome can be selected for using drug concentrations at the lower end of the indicated spectrum, while cancerous organoids might require higher concentrations.

Selection in Case of a Ubiquitously Expressed Resistance Cassette
Timing: 1-2 weeks 93. Keep organoids on selection medium for at least 1-2 passages to ensure proper selection.
Note: Organoids are most vulnerable at a single cell state which is why passaging organoids to single cells during the selection process is recommendable. Exceptions to this may be organoid lines with a poor single cell outgrowth efficiency.

Selection in Case of a STAR-Driven Resistance Cassette
Timing: 1-2 weeks  Note: We recommend to perform this step after FACS-purifying STAR + cells (step 98) to ensure that reporter-positive clones are picked.
Note: FACS purification of STAR + cells is suitable for all organoid lines in which STAR + cells constitute the stem cell population and therefore regenerate the organoid culture. It further ensures proper selection for cells having integrated the reporter which is particularly reassuring in cases of transient antibiotics selection. For designs in which a separate promoter drives the expression of a selection cassette, FACS purification of STAR + cells rules out partial silencing of the reporter at the STAR sites.
Note: When infecting mouse SI organoids, keep organoids on WENR medium during the selection process. Passaging mouse WENR organoids can be performed by mechanical fragmentation using stacked pipet tips (e.g., stack a p200 tip on top of a p1000 tip and vigorously pipet up and down). Subsequently, organoids can be reverted to a crypt-like state in a 2-step process: First, culture organoids for 2-7 days in medium consisting of 50% WENR and 50% ENR. As soon as Paneth cells have reformed (recognizable by their dark and granular appearance inside small budding structures on a bright field table-top microscope), the organoids can be maintained in ENR medium. As not all organoids revert at the same speed, it is advisable to carefully monitor the culture daily and base the timing of media change on the average organoid morphology. Different stages of reverting organoids are depicted in Figure 5.

Establishing a Clonal STAR Organoid Line
The outgrowth of isolated single cells into organoids is a highly inefficient process. Therefore, organoids need to be grown at a low-to-intermediate density prior to picking to ensure outgrowth while maintaining clonality. (A plating density of approximately 2,000-10,000 cells/100 mL Matrigel is recommended.) CRITICAL: The organoid density should be monitored carefully to ensure that no organoids are fusing during the outgrowth process. For organoid lines which are very sensitive to density, this process might involve plating cells at a density supporting single cell outgrowth and over the course of the next weeks replating intact organoid structures (without disruption) into larger volumes to ensure sufficient space for clonal organoid growth.
CRITICAL: The organoid should exceed a size of 300 cells prior to picking, to ensure successful passaging (approx. size depicted in Figure 4E, right panel). Growing the organoids as big as possible while ensuring their clonality and suitable culturing conditions will increase the success rate in establishing clonal organoid lines.
There are three time points at which a clonal organoid can be picked (ordered according to our preference):

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STAR Protocols 1, 100126, December 18, 2020 after selection on the initial plating Note: This option does not require dissociation to single cells. Therefore, it is very suitable for sensitive lines and can also be applied to mouse SI organoids which are growing in their regular crypt-like budding structure (ENR medium).
after selection and subsequent FACS purification of single STAR + cells Note: Broadly applicable strategy given that STAR + cells constitute the stem cell population, i.e., can regenerate the organoid culture.
after selection and subsequent thorough trypsinization to single cells.
Note: This option is preferable in organoid systems in which STAR + cells do not constitute the (sole) stem cell population. For instance, some lung and stomach cancers show upregulation of ASCL2, while it is not expressed during homeostasis (Hu et al., 2016;Huang et al., 2018;Zuo et al., 2018). Thus, the nature of STAR + cells in respective cancer organoids is not well defined yet.

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Note: Before you start: Label Eppendorf collection tubes and put them on ice Optional: Add 10 mL of Advanced DMEM/F12 into each collection tube to more easily release the organoids into medium.
Timing: 1 h (may be longer if multiple clones are picked) 100. Select a Matrigel droplet with a big STAR fluorescent organoid using a table-top microscope. 101. Discard culture medium of the selected culture well. 102. Use 50 mL ice-cold (4 C) Advanced DMEM/F12 to dissolve selected Matrigel droplet by tilting the culture plate and placing a pipette-tip (200 mL) above the droplet. 103. Take up the Matrigel droplet and transfer it into a 15 mL falcon tube. 104. Optional: Add 200 mL 13 dispase to the tube and incubate at 37 C for 5 min until Matrigel is properly dissolved. 105. Top up to 10 mL with ice-cold Advanced DMEM/F12 and carefully pellet organoids by spinning at 300 3 g at 4 C for 4 min. 106. Take off medium and carefully resuspend organoids in 100 mL Advanced DMEM/F12.
Note: If there are many organoids, increase the volume of Advanced DMEM/F12 to facilitate the next steps.
107. Spot organoid suspension onto the inside of the lid of a 10 cm culture dish, while making droplets of approximately 20 mL volume ( Figure 6C). 108. Locate organoids using a table-top or stereomicroscope. 109. For droplets having just one organoid, take up the medium droplet and transfer the organoid into a separate collection tube by carefully pipetting up and down. Verify that the organoid is transferred. If multiple organoids are in the same droplet, carefully take up single organoids using a 20 mL pipet, while checking through the oculars that the other organoids stay put. Transfer organoid into an Eppendorf tube ( Figure 6C). 110. Keep Eppendorf tubes on ice while picking. 111. Add 100 mL fresh Trypsin EDTA and incubate at 37 C for 5 min, while vortexing every minute.
Note: This step is recommended for most organoid lines as it allows for the highest plating density. Mouse SI organoids, however, and organoids with a low single cell plating efficiency may benefit from mechanical disruption instead.
Alternatives: (to step 114) Mechanically disrupt organoid by adding 200 mL cold Advanced DMEM and pipetting up and down with a p200 tip several times.
112. Check under the microscope that the organoids has dissociated into small fragments/single cells. Otherwise, prolong the trypsin incubation step. 113. Add 100 mL trypsin inhibitor and top up to 1 mL with Advanced DMEM. 114. Spin Eppendorf tube at 4 C, 500 3 g for 5 min. 115. Carefully take of supernatant, while leaving 5-10 mL supernatant to not agitate the pellet 118. Incubate the plate upside-down in the incubator for 10-25 min until the Matrigel has solidified 119. Add 250 mL organoid medium with 10 mM Y-27632 and 100 mg/mL primocin (since clonal organoids has been exposed to an environment outside the hood).
Note: In case of a low yield or organoid lines that are particularly sensitive to low density, it may be conceivable to plate STAR + cells together with reporter-negative cells to support organoid growth. FACS purification can be subsequently used to isolate all STAR + cells. Troubleshoot 7: Cell death after passaging clonal organoids Protocol EXPECTED OUTCOMES STAR activity reflects the transcriptional activity of the intestinal stem cell specific transcription factor ASCL2. Hence, STAR labels intestinal stem cells and, with decreasing intensity along the crypt-villus axis, transit amplifying cells (but not differentiated cells). The overall intensity levels depend on the number of ASCL2-binding motif repeats (STAR repeats, compare Figure 2), the brightness of the fluorophore, and the laser intensity used for exposure. While the nature of the STAR intensity gradient along the crypt-villus axis is independent of these parameters, the absolute intensity impacts on the visual manifestation of the gradient (Figures 6A and 6B). We therefore recommend analyzing multiple clonal organoids and compare their STAR expression to get the best fit for your research question.
1. Plate clonal STAR organoid cultures on glass for live-cell microscopy.
Note: It is convenient to have multiple clonal lines (> 3 clonal lines) to compare them with each other. In the end the aim is to report both reliable and efficient ASCL2 activity in stem cells.
2. Image STAR expression levels and pick your favorite. The range of STAR pattering in clonal organoids is depicted in Figures 6A and 6B.

LIMITATIONS
STAR reports the transcriptional activity of ASCL2, thus its application is limited to tissues that express ASCL2 (such as the small and large intestine, placenta, and several tumor tissues) (Jubb et al., 2006, The Human Protein Atlas). The recent discovery of ASCL2 controlling the LGR5 + stem cell state in the intestine, makes STAR a versatile tool to report intestinal stemness which partially overlaps but is more exclusive than Wnt activity (Murata et al., 2020;Oost et al., 2018;Schuijers et al., 2015). While STAR has been successfully applied in mouse and human intestinal tissues, extending the range to other species and tissues is hitherto untested. Furthermore, for tissues in which ASCL2 is expressed by a differentiated cell type rather than the stem cell compartment, reporter plasmids with STAR-driven expression of the antibiotic selection cassette are not suitable. It is advisable therefore to choose reporter plasmids with an independent, uniform promoter which drives the expression of a selection cassette and works in parallel to STAR (see Table 1).
When analyzing cell-type conversions, it is important to note that while the STAR signal is maintained by ongoing ASCL2 transcriptional activity, its decay is mainly determined by the half-life of the fluorescent protein itself and not directly related to the stability of ASCL2. Additionally, expansion media for organoids are generally rich in stem cell supporting factors which, in turn, can lead to widespread STAR activity at varying levels within a single organoid. For instance, with respect to the intestine, this reflects the stemness gradient from the crypt bottoms up to the transit amplifying zone. Please note, conditions that favor cellular differentiation, either by modulating the media composition or xenotransplantation in mice which provides a more native micro-environment, facilitates cellular diversity and might be desirable depending on the organoid system and the research question (Fumagalli et al., 2020;Oost et al., 2018).

TROUBLESHOOTING
Problem 1 STAR will not integrate into the genome.

Potential Solution for Lentiviral Approach (Protocol A)
Infecting organoids requires a much higher virus titre than most cell lines. Therefore, concentration of the virus prior to transduction and the addition of polybrene may be key to success. Potential Solution for Transposon-Based Approach with Electroporation (Protocol B) Double (or triple) the amount of STAR reporter DNA and of Tol2 transposase. When viability is not the limiting step, you may increase the poring pulse (up to 220 U in steps of 10 U for instance) and/or poring pulse length (up to10 ms, in steps of 1 ms).
Potential Solution for Transposon-Based Approach with Transfection Reagent (Protocol C) If transfection with a transfection reagent does not work well, consider using the transfection with electroporation protocol (protocol B).

Problem 2
No access to the indicated lentiviral envelope, packaging vectors, or transfection reagent.

Potential Solution
The three plasmids pHDM-G, pHDM-Hgpm2, and pRC-CMV-rev1b lead to the expression of VSV-G, gag and pol, and rev, respectively. Alternative plasmids containing these functional elements are commercially available, for instance on Addgene.
Alternatively to X-tremeGENE 9, the authors have successfully used TransIT-LT1 (Sopachem, MIR 2360) for the transfection of 293T cells during virus making (protocol A1, with a ratio of 2 mL transfection reagent for 1 mg of DNA). However, the transfection of STAR into organoids using a transfection reagent (protocol C2) has so far only been performed with X-tremeGENE 9 by the authors. While other transfection reagents might be suitable as well, we cannot comment on necessary adaptations to the existing protocol.

Problem 3
The cells die upon transduction with STAR lentiviruses.

Potential Solution
The virus titre might be too high. We suggest to set up a small dilution range of the virus (following ultracentrifugation) and transduce cells with different virus titres. If the cells are readily transducable, like some cell lines, ultracentrifugation is obsolete as the viral supernatant can be directly harvested of the virus-producing 293T cells, filtered with a 45 mm strainer (see protocol A step 9), supplemented with 8 mg/mL polybrene, and added on top of the receiving cells.

Problem 4
Electroporation kills all cells.

Potential Solution
Use up to 1 million cells per electroporation (while ensuring that the impedance is within the limits of 30-55 U). If still unsuccessful, lower the poring voltage and/or poring pulse length to make the procedure less harsh.

Problem 5
The cells are resistant to the selection antibiotic but there is no STAR signal.

Potential Solution
For plasmids in which the antibiotics selection cassette is controlled by an independent promoter, it is conceivable that expression of the reporter gene is compromised by epigenetic silencing of the ll OPEN ACCESS STAR promoter/enhancer site, while the ubiquitous promoter is still active. If this applies to a subculture, FACS purification of STAR + cells or establishing a clonal line from STAR + cells may solve the problem. Otherwise, when working with colorectal cancer organoids, make sure ASCL2 is still expressed in this tissue. You may also consider to integrate insulator sequences around the reporter to reduce the risk of epigenetic silencing (Ramezani et al., 2003). Note, however, that insulator sequences might be detrimental for the virus titre (Hanawa et al., 2009).

Problem 6
Loss of cells while picking clonal organoids.

Potential Solution
Pre-coat Eppendorf tubes with 10% fetal bovine serum or 1% BSA to prevent the cells from sticking to the plastic. If the cells easily stick to the pipet tip (check under the microscope), consider to precoat the tips as well. After trypsinizing the organoid and spinning down the single cell suspension, check under a table-top microscope to identify the location of the pellet. Carefully tilt the Eppendorf tube and take off media in multiple steps while checking that the pellet stays intact after each pipetting step. Collect supernatant in second Eppendorf tube to make sure the cells can be recovered in case the pellet is loose. Alternatively use 15 mL falcon tubes to get a cleaner pellet.

Problem 7
When generating clonal lines, the cells derived from a single organoid have difficulties surviving after the first passage.

Potential Solution
Organoid cells require a minimum density for successful outgrowth. Consider plating the cells in the presence of STAR-negative supporter cells of the same line to increase the plating density. This steps requires subsequent purification of STAR + cells at a later time-point (e.g., by FACS).

Problem 8
How to functionally test the STAR plasmid.

Potential Solution
Transiently transfect a cell line of your choice with the STAR plasmid. If the cells express ASCL2, the fluorescent protein should be visible under a confocal laser microscope. As 293T cells do not express ASCL2, STAR plasmids can be tested by co-transfecting ASCL2 (and oncogenic beta-catenin) to stimulate ASCL2 activity. For suitable plasmids, see Schuijers et al (Schuijers et al., 2015).

Problem 9
No access to growth factor conditioned media.
The recommended concentration of the Wnt Surrogate-Fc fusion protein (U-protein Express, Cat.No. #N001) is around 0.1-0.5 mM. It should be noted, however, that the optimal concentration might vary per organoid line. The recombinant Noggin and R-Spondin 3 may be used 1:100.
Other recombinant proteins are commercially available and might be suitable as well but the authors do not have any experience with them.

RESOURCE AVAILABILITY Lead Contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dr Hugo Snippert (H.J.G.Snippert@umcutrecht.nl).

Materials Availability
STAR plasmids referred to this study have been deposited to Addgene as listed in the Table 1. Additional plasmids required to perform the protocols are commercially available (Addgene or plasmID) and are listed in the Key Resources Table. Data and Code Availability The sequences of the STAR plasmids and the Tol2 transposase are available on Addgene via the Addgene numbers indicated in Table 1 and the Key Resources Table.