CAMBRIDGE BIONASH GROUP - European Bioinformatics Institute

Single-cell transcriptome analysis of bone-marrow residing human megakaryocytes
Iain Macaulay1, Willem H Ouwehand2, Sarah Teichmann1
1
Wellcome Trust Sanger Institute-European Bioinformatics Institute Single-Cell
Genomics laboratory, Welcome Trust Genome Campus, Hinxton, Cambridge
2
Department of Haematology, University of Cambridge and NHS Blood and
Transplant, Cambridge Biomedical Campus, Cambridge
Email: [email protected]
Group website: www.ebi.ac.uk/research/teichmann
Platelets are the second most abundant cell of the blood and a major
constituent of thrombi. At places of vascular damage and plaque rupture platelets
become activated, adhere to the damaged endothelium and drive thrombus
formation, directly contributing to acute heart attacks (AHA) and thrombotic stroke
(TS). Current pharmacological therapies focus at the molecular level, reducing their
activation and aggregation. While these therapies have enormously improved
outcome, particularly AHA resistance is a recognised problem and most platelet
inhibitors are ineffective in the management of TS.
There is a body of
epidemiological evidence, which shows that increased volume and activity of
platelets are risk factors of AHA and of poor outcome in the post-event setting.
Platelets are delivered to the blood by bone-marrow residing megakaryocytes
(MKs). MKs are derived from haematopoietic stem cells (HSCs) and during the final
stages of their differentiation they become polyploid. Endomitosis increases
chromosome content to levels ranging between 4N and 128N and concomitantly the
volume of cytoplasm increases and mature MKs are therefore extremely large (150400 𝞵m). MKs are rare and constitute only 0.01 and 0.05% of the cells in a bone
marrow harvest. The MK, and its erythroid equivalent, the erythroblast (EB), are
thought to be derived from the bipotent MK-EB progenitor or MEP.1-4 Recent studies
suggest however that the HSC may commit to the MK lineage in a more linear
fashion and this notion has become far more credible by our recently completed
study in transgenic mice.5
MKs can also be generated in the laboratory by culturing HSCs in the
presence of thrombopoietin and interkeukin 1β1-3, but high levels of ploidy remain
elusive for MKs obtained in vitro. We postulate that the MKs generated in vitro do
not reach the level of maturation, which is achieved if the HSC differentiates into MKs
in the bone marrow niche1-3. This assumption is supported by the results from our
array-based expression study, which identified a substantial number of transcripts
present in blood platelets at high levels but absent from in vitro generated MKs.
The aim of the proposed project is therefore to more accurately define the
transcript landscape of primary MKs and for reasons of comparison of primary EBs.
Research Ethics Committee permission has been secured for obtaining samples of
bone marrow from the hip bones of AHA patients or of healthy allogeneic blood stem
cell donors and from the sternum of patients undergoing elective cardiothoracic
procedures. EBs and MKs obtained by culture of CD34+ immune-purified stem cells
from cord and adult blood or by forward programming of human induced pluripotent
stem cells (iPSC)6 will be used to contrast their transcriptome landscapes with the
ones of primary progenitor cells. We are in the unique position to have access to
bone marrow samples from up to 80 AHA patients because of the BAMI study, a
phase III randomised controlled trial evaluating the effects of stem cell intracoronary
reinfusion on post-AHA heart tissue regeneration. Bone marrow will be harvested
three days after presentation with the heart attack and sternum harvests are obtained
from stable patients undergoing ‘heart’ surgery requiring medium sternotomy.
EBs and MKs are obtained from the harvested bone marrow samples or from
culture flasks. Because of the rarity of MKs, the aspirate is enriched by a Percoll
density gradient centrifugation. Following centrifugation the cells from the lower
interface are collected, washed and pelleted and a CD42b antibody separation kit
(EasySepTMPE positive selection kit, StemcellTM Technologies) is used to enrich the
MKs further. EBs are abundant in bone marrow aspirates and their enrichment is
therefore not required. Individual EBs and MKs are picked under 40x objective
inverted light microscope using a glass pipette mounted in a micromanipulator and
deposited into lysis buffer (0.2% triton, 1U/µL RNAse inhibitor) and stored at -80°C
until RNA-seq libraries are prepared. The wet-lab experiments are performed by
Fizzah Choudry, a MRC-funded clinical PhD fellow. She has already confirmed the
feasibility of the outlined approach and up to 2,000 MKs can be isolated from a 1 ml
bone marrow aspirate (5 ml aspirates are routinely obtained with REC permission)
and the BRC-EBI fellow will be responsible for the analysis.
Fizzah has under supervision of Dr Macaulay used the platform for single-cell
RNA sequencing (RNA-seq) currently operational at the WTSI-EBI single-cell
genomics centre.7 RNA will be harvested from at least 96 cells per harvest using
three biological replicates for EBs and MKs from the seven different biological
sources (hip harvest from AHA cases and healthy controls, sternum harvest from
‘elective’ surgery cases, cord and adult blood HSC-derived, forward programmed
iPSCs). For hip & sternum samples genomic DNA will be sequenced in addition to
the RNA to determine for the first time the level of haplotype silencing by imprinting in
polyploid (4N–128N) MKs. Coding single nucleotide variants will inform the
imprinting analysis for about 70% of genes at the planned number of samples to be
included in our experiment (this is under the assumption that the nature of imprinting
is independent of the hip vs sternum niche types and health vs ill-health status of
research volunteers).
Single cell RNA-seq, including downstream bioinformatics analysis, is well
established in the Teichmann group. They have recently collaborated on a
quantitative assessment of technical noise in single cell RNA-seq experiments using
the ERCC92 spike-in kit of mRNA standards.8 This technology was crucial in their
recent work describing a T helper cell subtype that synthesizes steroids de novo,
because the single cell transcriptome data revealed a cell surface marker for this Th2
cell population. This provided a means of purifying these cells and characterising
their suppressive function in T and B cell co-culture experiments.9
Applying this type of approach to MKs will increase our understanding the
basic molecular aspects of differentiation and maturation of primary MKs. This is
relevant because of the causative role of platelets in heart attack and thrombotic
stroke and the large unmet medical need in stroke management, where the current
platelet drugs do not work. The study will complement our recently completed RNAseq study of the eight classical types of blood progenitors, including EBs and MKs,
which for the first time revealed extensive alternative splicing at branch points in
haematopoiesis.10 In addition the study will answer the following basic cell biology
questions: i) is there in humans a subset of transcriptionally MK-primed HSCs, which
have recently been identified by us in a transgenic mouse model5, ii) does the level of
MK polyploidisation equates with their maturation; this important question continues
to be disputed in haematopoiesis community and iii) how does the transcriptome of
mature MKs poised to proplatelet formation differs from the MKs that do not show
evidence of proplatelet formation. In aggregate the outlined study does not only
address questions relevant for the understanding of the pathobiological processes
underlining the Number 1 killer in Western society, but also provides key insights in
the formation of the MK and its progeny the platelet. The latter is important for the
successful delivery of our NIHR, MRC and Wellcome Trust funded regenerative
medicine research, which aims to generate platelets for clinical use from MKs
obtained from human iPSCs.
References
1. Comparative gene expression profiling of in vitro differentiated megakaryocytes
and erythroblasts identifies novel activatory and inhibitory platelet membrane
proteins. Macaulay IC1, Tijssen MR et al. Blood. 2007;109:3260-9.
2. Genome-wide analysis of simultaneous GATA1/2, RUNX1, FLI1, and SCL
binding in megakaryocytes identifies hematopoietic regulators. Tijssen MR,
Cvejic A, et al. Dev Cell. 2011;20:597-609.
3. New gene functions in megakaryopoiesis and platelet formation. Gieger C,
Radhakrishnan A, Cvejic A, et al. Nature. 2011;480:201-8.
4. Compound inheritance of a low-frequency regulatory SNP and a rare null
mutation in exon-junction complex subunit RBM8A causes TAR syndrome.
Albers CA, Paul DS, et al. Nat Genet. 2012;44:435-9.
5. Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell
hierarchy, Sanjuan-Pla A, Macaulay IC, et al. Nature. 2013;502:232-6.
6. Generation of mature megakaryocytes from pluripotent stem cells by a
chemically-defined transcription factor-based forward programming approach.
Morreau T, Evans A, et al. Nat Methods, 2014; submitted
7. Single-cell paired-end genome sequencing reveals structural variation per cell
cycle. Voet T1, Kumar P, et al. Nucleic Acids Res. 2013;41:6119-38.
8. Accounting for technical noise in single-cell RNA-seq experiments. Brennecke P,
Anders S, et al. Nat Methods. 2013;10:1093-5.
9. Single cell RNA-sequencing reveals T helper cells synthesizing steroids de novo
to contribute to immune homeostasis. Mahata B, Zhang X, et al. Cell Reports.
2014;7:1130-42.
10. Transcriptome analysis of human hematopoietic progenitor cells reveals
extensive alternative splicing at lineage commitment. Chen L, Kostadima M, et al.
Science. 2014; accepted pending minor revisions.

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