CloneSeq: A highly sensitive analysis platform for the characterization of 3D-cultured single-cell-derived clones

Danny Bavli, Xue Sun, Chen Kozulin, Dena Ennis, Alex Motzik, Alva Biran, Shlomi Brielle, Adi Alajem, Eran Meshorer, Amnon Buxboim*, Oren Ram*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

Single-cell assays have revealed the importance of heterogeneity in many biological systems. However, limited sensitivity is a major hurdle for uncovering cellular variation. To overcome it, we developed CloneSeq, combining clonal expansion inside 3D hydrogel spheres and droplet-based RNA sequencing (RNA-seq). We show that clonal cells maintain similar transcriptional profiles and cell states. CloneSeq of lung cancer cells revealed cancer-specific subpopulations, including cancer stem-like cells, that were not revealed by scRNA-seq. Clonal expansion within 3D soft microenvironments supported cellular stemness of embryonic stem cells (ESCs) even without pluripotent media, and it improved epigenetic reprogramming efficiency of mouse embryonic fibroblasts. CloneSeq of ESCs revealed that the differentiation decision is made early during Oct4 downregulation and is maintained during early clonal expansion. Together, we show CloneSeq can be adapted to different biological systems to discover rare subpopulations by leveraging the enhanced sensitivity within clones.

Original languageAmerican English
Pages (from-to)1804-1817.e7
JournalDevelopmental Cell
Volume56
Issue number12
DOIs
StatePublished - 21 Jun 2021

Bibliographical note

Funding Information:
O.R. is supported by research grants from the European Research Council (ERC, # 715260 SC-EpiCode ), the Israeli Center of Research Excellence (I-CORE) program, the Israel Science Foundation (ISF, #1618/16 ), and Azrieli Foundation Scholar Program for Distinguished Junior Faculty. O.R. and A.A. are supported by Nofar (65883) of the Israel Innovation authority. E.M. is the Arthur Gutterman Family Chair in stem cell research and is supported by the Israel Science Foundation ( ISF1140/17 ). This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no 765966 – EpiSyStem.

Funding Information:
Overall, we sequenced 1,344 cells treated with Dox for 24 h. For differentiated cells, we collected 1,189 single cells from 3D hydrogels (HydrogelSC) and 536 clones (HydrogelCl). The analysis after 4 days of differentiation showed distinct clusters. Although CloneSeq provided superior coverage compared with single-cell assays, clones and single cells clustered together (Figure 6B). This strongly supports that the differentiation decision was made during the first 24 h upon downregulation of Oct4 and that clones merely amplified the differentiation signal of each single cell. Furthermore, clones and single cells were divided into three subpopulations based on marker genes that support the formation of ectoderm, endoderm, and mesoderm cellular states. Single cells and clones were equally distributed in the different subpopulations, with marker genes of endoderm such as Psap (Nakazawa et al., 2011), Crxos (Saito et al., 2010), Slc39a4(Zip4) (Dufner-Beattie et al., 2003), Klf5 (Moore-Scott et al., 2007), Dab2, and Gata6 (Morrisey et al., 1998) and of ectoderm such as Ssbp3 (Liu et al., 2016), F11r(JAM1) (Thomas et al., 2004), Hmga2 (Navarra et al., 2016), Lin28a (Parisi et al., 2017), and Id3 (Kowanetz et al., 2004; Kee and Bronner-Fraser, 2005). The main mesodermal marker genes that were upregulated are Gata2 (Orkin, 1992; Johnson et al., 2012), Sin3b (David et al., 2008), Irx3 (Mahlapuu et al., 2001), Prss8 (Sherwood et al., 2007; Popowski et al., 2017), and Arid3a (Tucker, 2017; Figures S6A?S6D). To further validate the specificity of our results, we performed an in silico test in which we summed up the expression profiles of ten randomly picked single cells without repeats and produce 118 pseudo-clones. The pseudo-clones show an averaged signal with no subpopulation structures (Figures S6E and S6F).O.R. is supported by research grants from the European Research Council (ERC, # 715260 SC-EpiCode), the Israeli Center of Research Excellence (I-CORE) program, the Israel Science Foundation (ISF, #1618/16), and Azrieli Foundation Scholar Program for Distinguished Junior Faculty. O.R. and A.A. are supported by Nofar (65883) of the Israel Innovation authority. E.M. is the Arthur Gutterman Family Chair in stem cell research and is supported by the Israel Science Foundation (ISF1140/17). This project has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Sk?odowska-Curie grant agreement no 765966 ? EpiSyStem. D.B. X.S. C.K. D.E. E.M. A.B. and O.R. conceived the study, prepared the figures, and wrote the manuscript. D.B. X.S. E.M. A.B. and O.R. designed the experiments. C.K. A.A. A.M. A.B. and D.E. performed PC9, ESCs, and IPSCs tissue culture and library preparation. S.B. and D.B. preformed microscopy and pipet aspiration test. D.B. and X.S. prepared the microfluidics system and performed scRNA-seq and CloneSeq experiments. X.S. and C.K. preformed clonal barcoding experiments. X.S. D.E. and O.R. preformed the computational analysis. The authors declare no competing interests.

Publisher Copyright:
© 2021 Elsevier Inc.

Keywords

  • 3D culturing
  • CloneSeq technology
  • cancer clonal expansion
  • cancer heterogeneity
  • cellular stemness
  • clone-to-clone variation
  • drop-based microfluidics
  • early differentiation
  • embryonic stem cells
  • single-cell RNA-seq

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