Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Summary

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Abstract

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Introduction

Most higher eukaryote populations are heterogeneous thus with analysis of pooled population, it is often difficult to interpret their cellular features. Individual cells within a population may be subtly different, and these differences can have important consequences for the property and function of the entire population^1,2. Especially, human embryonic stem cells (hESCs) are known to be heterogeneous, which causes different levels of pluripotency and diverse potentials to lineage specification in delicately distinctive ways^3,4. For example, different cell surface antigens can be used to categorize undi....

Protocol

1. Preparation of a 96-well Plate

1. Mix 1 μl of Single Cell DNase1 to 9 μl Single Cell Lysis Solution.
2. Put the 10 μl mixed solution in each well of 96-well PCR plate.

2. Detaching hESCs for FACS Purification

1. Detach OCT4::EGFP ES cell line from the 60 mm dish with 1 ml Accutase for 20 min, at 37 °C, which were neutralized with human ES media.
2. Prepare cell population in 1 ml FACS buffer and adjust the cell to 1 x 10⁶ cells/ml.
3. Pass the cell sample through a 35 μm cell strainer cap tube.
4. Store the tube in ice before cell sorting.

Results

Efficient and robust single cell RNA amplification

To minimize the transcriptional variation among hESCs, we used OCT4::EGFP hESC clone for FACS purification. After sorting OCT4::EGFP positive cells into a 96-well plate, each cell is lysed in lysis buffer and converted poly(A)+ RNA to full length cDNA using SMA-T15 (GACATGTATCCGGATGTTTTTTTTTTTTTTTT) primer and anchoring with SMA-A (ACATGTATCCGGATGTGGG) by using SMART template switching technology. The excess oligonuc.......

Discussion

Single cell gene profiling could be a major tool to predict functionality of a single cell or an entire population. Due to technical limitation, whole gene profiling analysis has been restricted to population averages. Variations in gene expression patterns and levels between individual cells and the subpopulations have been proposed to cause erroneous interpretation. Such diverse cellular aspects can be found in hESCs and their heterogeneity causes subtly different ability for maintaining pluripotency and fate specifica.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96-well PCR Plate	USA scientific	1402-8900
Ambion Cell Lysis Kit	Life Technologies	4458235
SMARTScribe Reverse Transcriptase	Clonetech	639536
ExoSAP-IT	USB	78200
Platinum Taq DNA polymerase High Fidelity	Invitrogen	11304
10 mM dNTP Mix, PCR Grade	Invitrogen	18427
SYBR Universal 2X Master Mix	Kapa biosystem	KR0389
Accutase	Innovative Cell Tech	S-1100-1
FACS buffer			45 ml PBS, 5 ml a-MEM, 100 μl DNase, filter sterilized
35 μm cell strainer cap tubes	BD Biosciences	352235