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 undifferentiated pluripotent stem cells,⁵ and the Austin Smith group proposed different levels of pluripotency in mouse embryonic stem cells, based on their morphology, differentiation propensity and dependency of signaling pathway⁶. This phenomenon was hypothesized in human embryonic stem cells⁷. Whereas the overall studies were performed among different stem cell lines, not individual single stem cells, it could be very intriguing to analyze different levels of pluripotency at the single cell level, which potentially affects their differentiation capacities toward all somatic cell lineages.

Cellular and molecular heterogeneity could be dictated by transcription profiling, which is called the ‘single cell transcriptome’ and emphasizes new approaches for quantifying gene expression levels^8-10. For analysis of gene expression levels in individual cells, we developed a simple, but robust protocol of single cell quantitative RT-PCR. We confirmed the efficacy and feasibility of our protocol by comparing each half of single cell lysates as well as serially diluted total RNAs of hESCs, resulting in minimal technical variations and differences. Further, we used a genetic reporter line to isolate homogenous population of hESCs using gene targeting system.  The donor vector for targeting OCT4 locus (OCT4-2A-EGFP-PGK-Puro construct) and a pair of TALEN plasmids were used¹¹. The donor vector and a pair of TALEN plasmids were introduced into hESCs (H9, WA09) using our nucleofection and clonal selection protocol and maintenance of hESCs was performed based on our routine protocol¹². We confirmed this genetic reporter line express EGFP for OCT4 expression in OCT4::EGFP hESCs.

Our result demonstrates that individual hESCs (sorted by OCT4::EGFP strongly positive cells) hold high levels of OCT4 expression, but different levels of NANOG expression. So, our single cell gene expression assay should be useful to study population heterogeneities of pluripotent stem cells.

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.

3. Lysis of FACS-purified Single Cell in Each Well of the 96-well Plate

1. Sort the sample for EGFP positive cells on a cell sorter with a trained operator. Put the single cell into Single Cell Lysis/DNase1 solution in 96-well PCR plate. If necessary, the 96-well plate with sample can be stored in a -80 °C deep freezer less than one month.
2. Incubate samples 5 min at RT for cell lysis.
3. Add 1 μl of Stop Solution to stop lysis reaction.
4. Incubate 2 min at RT.

4. Reverse Transcription

1. Add to each a 0.5 μl aliquot of 20 μM SMA-T15, SMA-A.
2. Add 4 μl 5X buffer, 2 μl DTT, 1 μl Reverse Transcriptase, and 1 μl dNTP to each well.
3. Perform reverse transcription in a thermal cycler.
   1. Set the thermal program at 42 °C × 90 min and inactivate Reverse Transcriptase at 85 °C × 5 min.

5. Amplification

1. Add 4 μl of ExoSAP-IT reagent to each reverse transcribed sample.
   1. Incubate samples at 37 °C for 15 min and 80 °C for 15 min to inactivate the ExoSAP-IT reagent.
2. Prepare PCR reaction mix with SMA-p2 (2 nM)
3. Add 10 μl of PCR reaction mix to each reverse transcribed sample.
4. Perform the amplification, consisting of 20 cycles of denaturation (94 °C for 30 sec), annealing (57 °C for 30 sec), and extension (68 °C for 10 min).

6. qRT-PCR Performance

1. Add 10 μl of 2X SYBR Green PCR Master Mix, 1 μl amplified cDNA, 2 nM primers, and 7 μl water to each well.
   1. Set the program followed by 95 °C for 3 sec, 60 °C for 30 sec x 40 cycles.
2. Perform in duplicate for technical errors.

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