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. 2022 Jul 19;3(3):101612.
doi: 10.1016/j.xpro.2022.101612. eCollection 2022 Sep 16.

Using human iPSC-derived kidney organoids to decipher SARS-CoV-2 pathology on single cell level

Katharina C Reimer  1 , Jitske Jansen  2 , Gijs J Overheul  3 , Pascal Miesen  3 , Ronald P van Rij  3 , Sergio H Triana  4 , Bart Smeets  5 , Rebekka K Schneider  6 , Rafael Kramann  7
Affiliations

Using human iPSC-derived kidney organoids to decipher SARS-CoV-2 pathology on single cell level

Katharina C Reimer et al. STAR Protoc. .

Abstract

We describe a protocol for single-cell RNA sequencing of SARS-CoV-2-infected human induced pluripotent stem cell (iPSC)-derived kidney organoids. After inoculation of kidney organoids with virus, we use mechanical and enzymatic disruption to obtain single cell suspensions. Next, we process the organoid-derived cells into sequencing-ready SARS-CoV-2-targeted libraries. Subsequent sequencing analysis reveals changes in kidney cells after virus infection. The protocol was designed for kidney organoids cultured in a 6-well transwell format but can be adapted to organoids with different organ backgrounds. For complete details on the use and execution of this protocol, please refer to Jansen et al. (2022).

Keywords: Cell biology; Microbiology; Organoids; Single cell; Stem cells.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Overview of the workflow from iPSC-derived kidney organoids to a targeted sequencing-ready single cell library
Figure 2
Figure 2
SARS-CoV-2 inoculation of iPSC-derived kidney organoids cultured on transwell filters (A) iPSC-derived kidney organoids cultured in a 6-well transwell plate in top and side view (air/liquid interface culture). (B) Control and SARS-CoV-2 conditions with organoids inoculated with mock medium (control) and with SARS-CoV-2 below and on top of the filter membrane (liquid culture). (C) Continued incubation of kidney organoids after 24 h inoculation in an air/liquid culture.
Figure 3
Figure 3
Sequential steps during organoid dissociation (A) iPSC-derived kidney organoids cultured in E6 medium in a 6-well format transwell system prior to dissociation. (B) iPSC-derived kidney organoids after three times washing with PBS. (C) Adding Digestion Solution on top of the transwell filter and resuspending organoids. (D) Mechanically dissociating organoids with a wide-bore tip. (E) Single cell suspension after transfer into a new 6-well plate. (F) Using a cell strainer to obtain a single cell suspension. Some non-digested extracellular matrix might be retained in the strainer. (G and H) Trypan blue stained single cell suspension of iPSC-derived kidney organoids at (G) 4× magnification, scale bar 250 μm and (H) 10× magnification, scale bar 125 μm, respectively in a Neubauer counting chamber. Note the low number of trypan blue positive cells.
Figure 4
Figure 4
PCR workflow to generate a SARS-CoV-2-targeted library and expected bioanalyzer traces of the constructed sequencing-ready libraries (A) PCR workflow to illustrate added adapters and target sequences during PCR 1–3. Inner: SARS-CoV-2 inner primer, outer: SARS-CoV-2 outer primer, N7XX: Illumina sequencing adapter (including P7 and i7). (B) Exemplary bioanalyzer trace of a standard 10× genomics Next GEM version 3.1 library. (C) Exemplary bioanalyzer trace of a SARS-CoV-2-targeted library after finishing PCR 3.
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