An Approach to Study Shape-Dependent Transcriptomics at a Single Cell Level

J Vis Exp. 2020 Nov 2:(165). doi: 10.3791/61577.

Abstract

Different types of cardiac hypertrophy have been associated with an increased volume of cardiac myocytes (CMs), along with changes in CM morphology. While the effects of cell volume on gene expression are well known, the effects of cell shape are not well understood. This paper describes a method that has been designed to systematically analyze the effects of CM morphology on gene expression. It details the development of a novel single-cell trapping strategy that is then followed by single-cell mRNA sequencing. A micropatterned chip has also been designed, which contains 3000 rectangular-shaped fibronectin micropatterns. This makes it possible to grow CMs in distinct length:width aspect ratios (AR), corresponding to different types of heart failure (HF). The paper also describes a protocol that has been designed to pick up single cells from their pattern, using a semi-automated micro-pipetting cell picker, and individually inject them into a separate lysis buffer. This has made it possible to profile the transcriptomes of single CMs with defined geometrical morphotypes and characterize them according to a range of normal or pathological conditions: hypertrophic cardiomyopathy (HCM) or afterload/concentric versus dilated cardiomyopathy (DCM) or preload/eccentric. In summary, this paper presents methods for growing CMs with different shapes, which represent different pathologies, and sorting these adherent CMs based on their morphology at a single-cell level. The proposed platform provides a novel approach to high throughput and drug screening for different types of HF.

Publication types

  • Video-Audio Media

MeSH terms

  • Animals
  • Animals, Newborn
  • Cell Adhesion
  • Cell Shape / genetics*
  • DNA, Complementary / genetics
  • Fibronectins / metabolism
  • Induced Pluripotent Stem Cells / metabolism
  • Myocytes, Cardiac / metabolism
  • Rats
  • Sarcomeres / metabolism
  • Single-Cell Analysis*
  • Transcriptome / genetics*

Substances

  • DNA, Complementary
  • Fibronectins