single cell

1. Single-cell technologies — studying rheumatic diseases one
cell at a time
Ritasman Baisya
4.7.2020

2. Let we can go in
reverse order……
From multicellular
to single cell level
…..

3. Points of discussion
• Ome & Omics
• Single cell technology – introduction
• Types
• Single cell In rheumatology
• ScRNA sequencing
• Single cell proteomics
• Single cell epigenomics
• Single cell in RA and SLE
• COVID immune profiling

4. Ome & omics
• ome as used in molecular biology refers to a totality of some sort
• Omics aims at the collective characterization and quantification of pools of biological
molecules that translate into the structure, function, and dynamics of an organism or
organisms.

6. Studying omics …..

7. Single cell technology
• Single-cell sequencing technologies refer to the sequencing of a single-cell genome
or transcriptome, so as to obtain genomic, transcriptome or other multi-omics
information to reveal cell population differences and cellular evolutionary
relationships.
Advantages -
• Detecting heterogeneity among individual cells
• Distinguishing a small number of cells, and
• Delineating cell maps

8. Single cell can describe OMICs

9. Timeline showing the development of technologies for single-cell
studies

10. Why single cell ?

11. Clinical applications of single-cell sequencing in different fields

12. Types
A. Single cell genomics - 1.Targeted-Genome Analysis ( SCGA , PACS )
2.Whole-Genome Analysis ( DOP-PCR , MDA,
SiC-Seq).
B. Single-Cell Epigenomics- 1. DNA methylation
2.Histone modification ( Drop Chip)
3.Chromatin assembly (dscATAC -seq )
C. Single cell transcriptomics – 1.Whole-Transcriptome Profiling
(inDrop ,Drop-seq, Hi-SCL )
2. Immune profiling

13. Types
• Single cell proteomics – 1.Mass cytometry ( CyTOF )
2. Imaging based proteomics
3.Membrane protein ( CITE seq , REAP seq ,Absec )
4.secreted protein ( cytokine and antibody secretion )
• Single cell metabolomics – 1. Detection of Cancer Cells (Lactate Secretion)
2. Screening of Bacterial Mutant Libraries (Ethanol or
Lactate Secretion)

14. Characteristics of cell isolation technology
• Efficiency or throughput (how many cells can be isolated in a certain time)
• Purity (the fraction of the target cells collected after the separation)
• Recovery (the fraction of the target cells obtained after the separation as
compared to initially available target cells in the sample).

15. Two types isolation technique
• Physical properties based - size, density, electric changes, and deformability (
density gradient centrifugation, membrane filtration and microchip-based capture
platforms )
• Cellular biological characteristics based comprising of affinity methods, such
as affinity solid matrix (beads, plates, fibers), fluorescence-activated cell sorting,
and magnetic-activated cell sorting

16. Single cell in rheumatology
• Better understanding of the heterogeneity of human cells
• Pathogenic mechanisms of rheumatic diseases at different omics levels with
therapeutic target
• Single-cell RNA sequencing facilitates the simultaneous interrogation of the
transcriptome of thousands of cells
• Transcript-based analyses of paired antigen receptor sequences and immune
profiling
• Mass cytometry enables the deep immunophenotyping and functional characterization
of protein markers
• Mass spectrometry imaging provide information on the spatial relationships between
molecules

17. Single cell in rheumatology ( pictorial form )

18. Single cell projects
• Several collaborative projects have been launched for single-
cell analyses in rheumatology
• The Accelerating Medicines Partnership (AMP) - RA and
SLE network has made several important discoveries at the
single- cell level
• It developed optimized protocols for the isolation of cells from
multiple tissues, including the synovium, alongside a protocol
for the cryopreservation of synovial tissue allowing for later
digestion
https://www.nih.gov/research-training/accelerating-medicines-partnership-amp

19. Individual Discussion with rheumatological application
• Single- cell RNA sequencing (scRNA- seq)
• Antigen receptor sequencing
• Mass cytometry
• Mass- spectrometry-based imaging
• Variety of epigenomic platforms
• Multi- omics technologies that enable simultaneous analyses of DNA,
RNA and protein markers

20. Gene expression analysis – ScRNA sequencing
• Steps – single cell isolation, reverse transcription, cDNA amplification & library
construction
• Additional scRNA-seq technologies that preserve the tissue context or subcellular
localization of transcripts have also been described
• Single-molecule RNA fluorescence in situ hybridization (FISH) & multiplexed error-
robust FISH (MERFISH) enable spatially resolved transcriptomic analyses of single
cells.
• InDrop system, droplet sequencing (Drop-seq), the Seq-Well platform and gene
expression cytometry

22. Technique Single cell isolation
method
Reverse transcription method cDNA
amplificati
on
Method
Throughput
( number of
cell
analyzed )
UMI
Tang et al Micropipetting Poly A based PCR 10
inDROP Droplet based
microfluidic system
Poly A based IVT 10^4
Drop - Seq Droplet based
microfluidic system
Poly A based PCR 10^4
Seq wells Microwells Poly A based PCR 10^ 4
Cyto Seq Microwells Poly A based PCR 10^5
Div Seq FACS Template switching PCR 10^3
DroNc seq Droplet based
microfluidic system
Template switching PCR 10^4
STRT Micropipetting Template switching PCR 10^2
CEL seq Micropipetting Poly A based PCR 10^2
SUPer-Seq Micropipetting Random primer based PCR 10^2

23. ScRNA - Role in rheumatology
• In renal tubular cells of SLE patients, number of interferon- inducible transcripts by scRNA-seq
correlate with histological evidence of chronicity, proteinuria and immunoglobulin deposition
• scRNA- seq study also found transcriptomically distinct subpopulations of fibroblasts in
synovial tissue samples from patients with RA or OA
• Presence of large numbers of the CD34−THY1+ subset in RA synovium is correlated with a
high amount of immune cell infiltration, a high Krenn synovitis score and joint hypertrophy.
• scRNA- seq has also been performed on fluorescence- activated cell sorting (FACS)-purified
natural antibody- producing CD27+IgD+ B cells, which are found in reduced numbers in
patients with RA

24. Paired antigen sequencing
• Several NGS-based platforms have been developed to profile the paired functional B cell receptor
(BCR) sequences of individual B cells
• Single-cell BCR sequencing of plasmablasts isolated from patients with RA identified BCRs specific
for cyclic citrullinated peptides and other RA- associated autoantigens
• Sequencing of plasmablasts from ACPA(+) individuals, who are at risk of developing RA, revealed
both IgA- and IgG- secreting clones responsive to common RA autoantigens, suggesting involvement
of mucosal immunity in the early stages of RA development
• In vitro expression of paired heavy- chain and light- chain sequences to construct clone- specific
immunoglobulins.

25. Schematic of single B cell capture, library construction and sequencing

26. T cell sequencing
• Established NGS-based single-cell methods exist to profile T cell receptor (TCR)
sequences with paired α-chains and β-chains
• Single-cell analysis preserves information on functional TCRs
• Autoreactive T cells isolated from inflamed tissues relevant to rheumatic diseases
such as synovial tissue, kidney and skin and to determine the antigen specificities of
T cells
• Dysregulation of specific T cell subsets directly contributes to tissue damage- TH17
cells are found in the peripheral blood of patients with SLE and kidneys of patients
with lupus nephritis

27. Single cell proteomics – mass cytometry
• Capturing proteomic information from single cells has proven to be a substantial
technical challenge.
• Mass cytometers require antibodies conjugated to rare earth metal isotopes of
defined atomic mass instead of fluorochromes.
• To quantify the expression level of proteins on individual cells, metal isotopes can
be conjugated to primary antibodies for mass cytometry analysis
• Mass cytometry has been widely used to perform immune monitoring of
phenotypic and functional protein markers in rheumatic diseases.

28. Basic of mass-cytometry
A. Antibodies conjugated to metal isotopes
B. Single-cell suspension carried into nebulizer
of the mass cytometer
C. Cells injected into an argon flow chamber are
exposed to a plasma torch
D. Metal tagged vaporized cells form ion clouds
sent through Quadrupole for purification and
concentration.
E. Times of flight (TOFs) of the ions from the
plasma are measured by a mass spectrometer
detector.
F.Individualized cells are profiled by atomic
mass (i.e. lighter metals arrive first)

29. Mass cytometry in rheumatological application
• Analyses of whole-blood in SLE treated with TLR ligands revealed differences in
signaling protein activation and downstream cytokine production profiles
• A molecular signature defined by increased CC-chemokine ligand 2 (CCL2), CCL4
and IL-1 receptor antagonist in CD14hi classical monocytes was found by mass
cytometry in patients with juvenile SLE
• Mass cytometry also facilitates the identification of unique immune cell subsets that
are aberrantly expanded in patients with rheumatic diseases

30. • Discovery of immune cell populations, such as PD1hiCXCR5−CD4+ T cells that
promote humoral immunity in inflamed RA synovial tissue & PD1+CD4+ T cells in
the salivary glands of patients with primary sjögren syndrome .
• CD27−HLA DR+ effector memory CD4+ T cell subset was discovered that
correlated with RA disease severity
• Deep immunophenotyping and functional characterization of the immune system
Mass cytometry in rheumatological application

31. Mass spectrometry imaging (MSI)
• Mass spectrometry imaging (MSI) is a technique used in mass spectrometry to visualize
the spatial distribution of molecules, as biomarkers, metabolites, peptides or
proteins by their molecular masses
• Most common ionization technologies - DESI imaging, MALDI imaging and
secondary ion mass spectrometry imaging (SIMS imaging)
• Two revolutionary MSI methods, multiplexed ion beam imaging (MIBI) & imaging
mass cytometry achieve highly multiplexed images of fixed tissue samples

33. Application of MSI in rheumatology
• Their potential to study the pathological functions of tissue-resident immune cells
and immune cell infiltrates in inflamed tissues in rheumatic diseases has not yet been
explored.
• Dysregulation of TRM cells has been implicated in the pathology of several
rheumatic diseases, including RA and juvenile idiopathic arthritis (JIA)
• It offers the opportunity to examine the functions of tissue- resident immune cells in
immunosurveillance and in the pathophysiology of immune- mediated diseases

34. Other single cell proteomics technology
• Single- cell western blotting enables the analysis of individual cells seeded in
microwells by thin-layer gel electrophoresis & photo-immobilization, followed by
antibody hybridization
• To dissect the heterogeneity of neural stem cells & to track their lineage
commitments during differentiation
• An established protocol exists for quantum dot-based immunohistochemistry
analysis, which can be applied to analyse formalin-fixed and paraffin-embedded
(FFPE) clinical samples for rheumatology research

35. A highly multiplexed cytometric imaging approach,
termed co-detection by indexing (CODEX), is used here to
create multiplexed datasets of normal and lupus
(MRL/lpr) murine spleens.
The fidelity of multiplexed spatial cytometry
demonstrated here allows for quantitative systemic
characterization of tissue architecture in normal and
clinically aberrant samples.

36. Other single cell proteomics technology
• Microengraving method has been used to interrogate single- cell variations in
cytokine secretion by human PBMCs in response to immune stimulation.
• single- cell barcode chip (SCBC) uses a microfluidic system to simultaneously
measure multiple molecules secreted from single cells and has been used to dissect
the functional heterogeneity of cytotoxic T cells

37. Single cell epigenomics
• The epigenome is highly dynamic and is regulated by internal and
external factors such as hormones, metabolites, microorganisms,
environmental factors and ageing.

38. DNA methylation
• The molecular links between DNA methylation and embryonic development,
genomic stability, transcription regulation, gene imprinting, repetitive DNA
silencing and X chromosome inactivation have been extensively studied
• DNA methylation at promoters is mainly associated with gene silencing, whereas
intragenic methylation at actively transcribed genes is essential for transcriptional
fidelity.
• Methylation at CpG sites is a classic epigenetic mechanism by which de novo
cytosine modifications can be stably transmitted through mitosis and meiosis and
between generation

39. Single-cell bisulfite sequencing (scBS-seq)
• The current gold standard for the detection of DNA methylation is bisulfite
sequencing
• Treatment of genomic DNA with sodium bisulfite converts unmethylated cytosines
to uracils, whereas methylated cytosines are resistant to this chemical modification.
• This approach essentially transforms epigenetic marks into a genetic code that can
be measured by microarray or NGS technology.
• Reduced representation bisulfite sequencing (RRBS), single- cell bisulfite
sequencing (scBS- seq), single- cell whole- genome bisulfite sequencing (WGBS)

41. Histone modification analysis
• Histone post- translational modifications ( histone marks) - important for
regulation of chromatin dynamics
• Essential for successful haematopoietic cell development, effective immune
responses against pathogens & the maintenance of immune tolerance
• A genetic mutation in V(D)J recombination- activating protein 2 (RAG2) that
abrogates its ability to bind histone H3 trimethylation at lysine 3 (H3K4me3)
marks results in inefficient V(D)J recombination and immuno deficiency
• Mis-writing, mis-erasing and mis-interpretation of histone post- translational
modifications are signatures of a wide variety of human diseases

42. Technology of histone modification
• ChIP- seq analysis of histone modifications at single- cell resolution has been
reported
• Eigenetic landscape profiling using cytometry by time- of-flight (EpiTOF)
takes advantage of the high multiplexing power, single- cell resolution and the
potential for quantitative measurement provided by mass cytometry to
simultaneously detect the bulk concentrations of a variety of histone marks in
individual cells

43. Chromatin structure analysis
• Invention of assay for transposase-accessible chromatin using sequencing (ATAC- seq)
has revolutionized the genome- wide analysis of chromatin structure
• Used to map the epigenomic landscapes of haematopoietic stem cells , progenitor cells and
has revealed a regulatory network that governs immune cell differentiation during
haematopoiesis
• CD4+CD28+KIR+CD11ahi cells & CD4+CD28+KIR−CD11alo cells purified from
patients with SLE were characterized by genome- wide increases in chromatin accessibility,
• CD4+CD28+ KIR+CD11ahi cells were enriched for differentially accessible regions in pro-
inflammatory genes

46. Single-cell multi-omics technologies
Several methods have been developed to conduct parallel analysis of either
genomic, transcriptomic and epigenomic data or genomic, transcriptomic and
proteomic data in single cells.

47. Parallel analyses of the genome, transcriptome and epigenome

48. Parallel analyses of the genome, transcriptome and epigenome

49. Parallel analyses of the genome, transcriptome and proteome
• PLA and proximity extension assay (PEA) simultaneously analyse
protein and RNA at single- cell resolution.

50. ScRNA technology in lupus nephritis
• Transcriptomic evaluation at the single-cell level helps to identify rare cell populations and evaluate
the communication between renal cell types and inflammatory leukocytes and fibroblast
• plate-based CEL-SEQ2 approach on the leukocytes sorted from the kidneys of LN patients reported
the immune landscape of the kidney- T follicular helper regulatory cells subsets
• Single-cell transcriptomic analysis revealed an interferon response signature that correlated with
clinical parameters, including histological features such as glomerular immunoglobulin G deposition
and chronicity
• Variable immune cell infiltration in same histology of lupus nephritis – predict B-cell depleting
approaches in patients with high numbers of kidney-infiltrating B cells
• early signs of extracellular matrix expression in tubular cells, even in the absence of other evidence
for fibrosis and chronicity, may suggest antifibrotic approaches

51. 1. Type I interferon (IFN)-response signatures in
tubular cells and keratinocytes distinguished
patients with LN from healthy control subject
2.
a high IFN response signature & fibrotic
signature in tubular cells were each associated
with failure to respond to treatment.
3. Analysis of tubular cells from patients with
proliferative, membranous and mixed LN
indicated pathways relevant to inflammation
and fibrosis, which offer insight into their
histologic differences

53. Single cell technology in RA
• Sequencing of 20,387 single cells reveals 13
transcriptomically distinct clusters
• These encompass an unsupervised draft atlas of the
autoimmune infiltrate that contribute to disease biology
• Identification of previously uncharacterized fibroblast
subpopulations and discern their spatial location within the
synovium.
• CD55+ fibroblasts expressed functional modules associated
with endothelial cell proliferation and regulation of reactive
oxygen species responses,
• Both CD90+ fibroblast groups were enriched for modules
associated with metallopeptidase activity and the
organization of the extracellular matrix

55. What they have found ? Immune atlas
• Transcriptional changes is analysed in peripheral blood mononuclear cells during the
recovery stage of COVID-19 by single-cell RNA sequencing technique
• T cells decreased remarkably, monocytes increased in patients in the early recovery
stage (ERS) of COVID-19
• Increased ratio of classical CD14++ IL1β+ monocytes with high inflammatory gene
expression
• CD4+ T cells and CD8+ T cells decreased significantly and expressed high levels of
inflammatory genes in the ERS.
• the plasma cells increased remarkably, whereas the naïve B cells decreased

56. • Several novel B cell-receptor (BCR) changes were identified, such as IGHV3-23
and IGHV3-7
• IGHV3-23-IGHJ4, indicated a monoclonal state associated with SARS-CoV-2
specificity
• Monocytes may produce a larger number of inflammatory mediators, including IL-
1β and IL-6, contributing to inflammatory storm
• In LRS patients, both DCs-derived TNFSF13 and IL-18 and T cell-derived IL-2, IL-
4 may promote B cell survival, proliferation, and differentiation. Consequently, B
cells produce numerous SARS-COV-2- specific antibodies to clear viruses
What they have found ?

58. Take home points
• Analysis of individual cells – genomics , epigenomics , transcriptomics ,proteomics
• Area of interest in rheumatology – lupus nephritis and RA
• AMP- SLE and RA network are actively working in single cell technology
• ScRNA sequencing is most commonly used in rheumatology
• Others – mass cytometry , MSI , epigenomic platforms
• Helping in precision medicine and future therapy
• Parallel multi-omics technology – a boon
• Immune profiling helps to explore immunopathogenesis including COVID19

59. References
• Cheung, P., Khatri, P., Utz, P.J. et al. Single-cell technologies — studying rheumatic diseases one cell
at a time. Nat Rev Rheumatol 15, 340–354 (2019)
• Wen, W., Su, W., Tang, H. et al. Immune cell profiling of COVID-19 patients in the recovery stage
by single-cell sequencing. Cell Discov 6, 31 (2020).
• Der E, Suryawanshi H, Buyon J, Tuschl T, Putterman C. Single-cell RNA sequencing for the study
of lupus nephritis. Lupus Sci Med. 2019;6(1):e000329.
• Matuła, K., Rivello, F., Huck, W. T. S., Single‐Cell Analysis Using Droplet Microfluidics. Adv.
Biosys. 2020, 4, 1900188.
• Donlin LT, Park SH, Giannopoulou E, et al. Insights into rheumatic diseases from next-generation
sequencing. Nat Rev Rheumatol. 2019;15(6):327-339. doi:10.1038/s41584-019-0217-7
• Der, E., Suryawanshi, H., Morozov, P. et al. Tubular cell and keratinocyte single-cell transcriptomics
applied to lupus nephritis reveal type I IFN and fibrosis relevant pathways. Nat Immunol 20, 915–
927 (2019)
• Stephenson, W., Donlin, L.T., Butler, A. et al. Single-cell RNA-seq of rheumatoid arthritis synovial
tissue using low-cost microfluidic instrumentation. Nat Commun 9, 791 (2018).