Assessing Cardiac Reprogramming using High Content Imaging Analysis

Summary

We present a protocol to quantify directly reprogrammed induced cardiomyocyte-like cells (iCMs) in vitro using high content imaging analysis. This method allows us to quantify the efficiency of cardiac reprogramming in an automated manner and to directly visualize iCMs.

Abstract

The goal of this protocol is to describe a method for quantifying induced cardiomyocyte-like cells (iCMs), which are directly reprogrammed in vitro by a reprogramming technique. Cardiac reprogramming provides a strategy to generate new cardiomyocytes. By introducing core cardiogenic transcription factors into fibroblasts; fibroblasts can be converted to iCMs without transition through the pluripotent stem cell state. However, the conversion rate of fibroblasts to iCMs still remains low. Accordingly, there have been numerous additional approaches to enhance cardiac reprogramming efficiency. Most of these studies assessed cardiac reprogramming efficiency using flow cytometry, while at the same time performed immunocytochemistry to visualize iCMs. Thus, at least two separate sets of reprogramming experiments are required to demonstrate the success of iCM reprogramming. In contrast, automated high content imaging analysis will provide both quantification and qualification of iCM reprogramming with a relatively small number of cells. With this method, it is possible to directly assess the quantity and quality of iCMs with a single reprogramming experiment. This approach will be able to facilitate future cardiac reprogramming studies that require large-scale reprogramming experiments such as screening genetic or pharmacological factors for enhancing reprogramming efficiency. In addition, the application of high content imaging analysis protocol is not limited to cardiac reprogramming. It can be applied to reprogramming of other cell lineages as well as any immunostaining experiments which need both quantification and visualization of immunostained cells.

Introduction

Cardiac reprogramming has been developed as an alternative approach to stem cell mediated approaches to generate new cardiomyocytes. Given that it does not transition through stem cell state, it has a high potential to bypass some inherited limitations in stem cell mediated approaches. It has been shown that viral infection of at least three or four cardiogenic transcription factors into fibroblasts can convert fibroblasts toward a cardiac fate by eliminating fibroblast gene programs and rebuilding cardiogenic transcriptional networks in fibroblasts^{1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17}.

Since the first landmark study demonstrating cardiac reprogramming in vitro¹, the cardiac reprogramming protocol has been optimized by numerous studies^{3,5,6,7,9,11,12,13,14,15,16,18}. Common technical approaches to assess cardiac phenotypes in fibroblasts following cardiac reprogramming have been flow cytometry analysis for quantifying cells expressing specific cardiomyocyte markers and immunocytochemistry for visualizing those cells at a single cell level. Although both experiments (i.e., flow cytometry and immunocytochemistry) are to demonstrate expression of cardiomyocyte markers using the same antibodies, they have to be performed separately. In addition, flow cytometry needs a relatively larger number of cells, thereby increasing the amount of reagents needed for the experiment. Alternatively, cardiomyocyte marker positive cells can be quantified by manual counting following immunocytochemistry. However, it is very labor intensive and tends to be less accurate.

The purpose of this protocol is to describe the method that can quantify and visualize iCMs by a single immunostaining experiment using automated high content imaging analysis. It requires a relatively small number of starting cells because this protocol is performed in a well of 24-well plate. As many as three different markers can be used at the same time. Single, double, and triple positive cells can be automatically quantified. In addition to quantification of immunostained cells, high content imaging analysis provides high quality 2-100x objective images. If necessary, the same immunostained cells used in high content imaging analysis can be re-used for further imaging studies, such as confocal microscopy. The main advantage of this protocol is that it provides not only unbiased quantification of iCMs with a much smaller number of cells, but also visualization of iCMs. Furthermore, this protocol can be utilized for assessing non-cardiac lineage reprogramming (e.g., iPSC, neuron, and hepatocyte reprogramming).

Protocol

All animal procedures were performed with the approval of Vanderbilt University Medical Center Institutional Animal Care and Use Committee. 1. Retrovirus generation and in vitro cardiac reprogramming Culture Platinum E cells in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, 1 µg/mL puromycin, and 10 µg/mL blasticidin until Platinum E cell confluency reaches 70%–80%. On Day 1, seed ~0.55 x 106 cells (first well) and ~0.18 x 106<…

Representative Results

Following reprogramming experiments, we quantified iCMs using high content imaging analysis as described above. Composite images of 36 imaging sites that were used for high content imaging analysis were shown in Figure 1. iCMs are defined as double-positive cells (α-actinin+Titin-eGFP+) in these experiments. High content imaging analysis shows that ~26% of cells exhibited both cardiac markers following M-G-T-H transduction, while ~1% of empty vector transduced cont…

Discussion

The previous reprogramming studies assessed reprogramming efficiency using flow cytometry and demonstrated the structural quality of iCMs using immunocytochemistry in two separate experiments. Flow cytometry analysis requires a much larger number of starting cells, thereby increasing the scale of experiments. In contrast, high content imaging analysis can evaluate both quality and quantity of iCM reprogramming by a single experiment with a relatively small number of cells. Therefore, this new method can provide an effici…

Materials

A83-01	Tocris	2939
anti-chicken Alexa 488	Thermofisher	A11039
anti-GFP antibody	Invitrogen	A10262
anti-mouse Alexa 555	Thermofisher	A21422
anti-α-actinin antibody	Sigma	A7811
DAPI solution	Vector labs	H1200
Fugene 6	Promega	E2691
Insulin-Transferrin-SeleniumG supplement	Invitrogen	41400-045
Medium 199	Invitrogen	11150059
MEM vitamin solution	Invitrogen	11120-052
MetaXpress software	Molecular device
Micro XL automated cell imagining system	Molecular device
Minimal essential amino acid solution	Sigma	M7145
Opti-MEM	Gibco	31905-070
PES filter (0.45 µm)	Thomas scientific	1159T84
Platninum E cells	Cell Biolabs	RV-101
Polybrene	Sigma	H9268
SB431542	Sigma	S4317
Universal blocking buffer	BiogeneX	HK083-50K