Review Article

Spatially resolved transcriptomics:

advances and applications
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Honglin Duana, Tao Chenga,b,c,*, Hui Chenga,b,c,*

a
State Key Laboratory of Experimental Hematology, Haihe Laboratory of Cell Ecosystem, National Clinical Research Center
for Blood Diseases, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking
Union Medical College, Tianjin, China; bCenter for Stem Cell Medicine, Chinese Academy of Medical Sciences, Tianjin, China;
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c
Department of Stem Cell & Regenerative Medicine, Peking Union Medical College, Tianjin, China

Abstract
Spatial transcriptomics, which is capable of both measuring all gene activity in a tissue sample and mapping where this activity
occurs, is vastly improving our understanding of biological processes and disease. The field has expanded rapidly in recent
years, and the development of several new technologies has resulted in spatially resolved transcriptomics (SRT) becoming highly
multiplexed, high-resolution, and high-throughput. Here, we summarize and compare the major methods of SRT, including imaging-
based methods, sequencing-based methods, and in situ sequencing methods. We also highlight some typical applications of
SRT in neuroscience, cancer biology, developmental biology, and hematology. Finally, we discuss future possibilities for improving
spatially resolved transcriptomic methods and the expected applications of such methods, especially in the adult bone marrow,
anticipating that new developments will unlock the full potential of spatially resolved multi-omics in both biological research and
the clinic.
Key Words: In situ hybridization; In situ sequencing; Spatially resolved multi-omics; Spatially resolved transcriptomics

1. INTRODUCTION However, most single-cell RNA sequencing (scRNA-seq)

Omics technologies, which provide a comprehensive, global
assessment of a set of molecules,1 offer a novel approach for
the study of biological activity in life-sciences research. In recent
years, the omics field has advanced considerably due to devel-
opments in the high-throughput analysis of biological molecules.
Transcriptomic technologies in particular have provided rich
insights into cellular characteristics, and measure the complete set
of RNA transcripts produced by the genome under specific cir-
cumstances. Indeed, characterizing the transcriptome of individ-
ual cells is fundamental to improve our understanding of complex
biological systems. Over the last decade, the rapid development
of single-cell sequencing technology has greatly accelerated bio-
medical research, allowing scientists to overcome the major chal-
lenge of heterogeneity within biological samples and leading to
the publication of a series of single-cell transcriptome atlases.2–4
* Address correspondence: Hui Cheng, Tao Cheng, 288 Nanjing Road, Tianjin,
China. E-mail address: chenghui@ihcams.ac.cn (H. Cheng); chengtao@ihcams.
ac.cn (T. Cheng).
Conflict of interest: The authors declare that they have no conflict of interest.
This work was supported by grants from the Ministry of Science and Technology
of China (2021YFA1100900 and 2020YFE0203000), the National Natural Science
Foundation of China (81730006, 81922002, 81861148029, and 81870086),
CAMS Innovation Fund for Medical Sciences (2021-I2M-1-040 and 2021-I2M-1-
019), Haihe Laboratory of Cell Ecosystem Innovation Fund (HH22KYZX0016) and
Distinguished Young Scholars of Tianjin (19JCJQJC63400).
Blood Science (2023) 5, 1–14
Received June 28, 2022; Accepted October 19, 2022.
http://dx.doi.org/10.1097/BS9.0000000000000141
Copyright © 2022 The Authors. Published by Wolters Kluwer Health Inc., on
behalf of the Chinese Medical Association (CMA) and Institute of Hematology,
Chinese Academy of Medical Sciences & Peking Union Medical College
(IHCAMS). This is an open access article distributed under the Creative
Commons Attribution License 4.0 (CCBY), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is
properly cited.
www.blood-science.org 
approaches involve isolating cells from their original position,
meaning that spatial information is lost during transcriptome
profiling. This omission makes it impossible to examine how
a given cellular state interacts with neighboring cells or the
surrounding extracellular matrix.2,5,6 To make up for this defi-
cit, a new technology known as spatially resolved transcrip-
tomics (SRT) aims to determine gene expression profiles while
preserving information about the spatial context of the tissue.
This technique is of great significance, since identifying the
specific spatial location and organization of different cell types
in multicellular tissues or organs is the foundation for research
into cellular biological functions. Transcriptomics that is
accompanied by spatial information will undoubtedly enable
us to better understand the organization of cells and tissues,
and how this organization influences biological function.7 The
application of SRT has also opened up new areas of spatially
resolved omics, including genomic, transcriptomic, proteomic
and potentially other omic data with retained positional infor-
mation. The ability of SRT to change the way we understand
complex tissues earned it the title of Method of the Year 2020
in Nature Methods.8 In this review, we will first summarize
and compare the major methods of SRT (Table 1), highlight
some current applications of SRT, and finally discuss our per-
spectives on the potential future applications of this method.
2. METHODS AND ADVANCES
The core, underlying principle of SRT, involves the acquisition
of gene expression profiles while retaining spatial information.
Broadly speaking, SRT methods can be classified into 3 groups:
imaging-based methods, sequencing-based methods, and in
situ sequencing methods. Imaging-based methods are carried
out using a microscope, and the transcriptomes are read out
through fluorescence in situ hybridization (FISH). Sequencing-
based methods are performed using next-generation sequenc-
ing technology, and RNA with spatial information is captured
and subsequently sequenced. In situ sequencing methods are
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Table 1
Major SRT methods.
Category
smFISH
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Multiplexed FISH
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Sequencing-based
methods that do not
validate cell types
Sequencing-based
methods that
validate cell types
Sequencing-based
methods in vivo
In situ sequencing
methods
Methods for FFPE
tissue
corrFISH = correlation FISH, FFPE = formalin-fixed paraffin-embedded, FISH = fluorescence in situ hybridization, HDST = high-definition spatial transcriptomics, LCM = laser-capture microdissection,
MERFISH = multiplexed error-robust FISH, scRNA-seq = single-cell RNA-sequencing, smFISH = single-molecule FISH, seqFISH = sequential barcoded FISH, SRM = super-resolution microscopy, ST =
spatial transcriptomics, TIVA= transcriptome in vivo analysis, DBiT-seq =deterministic barcoding in tissue for spatial omics sequencing, ExFISH=expansion microscopy of fluorescence in situ hybridization,
FISSEQ=fluorescent in situ RNA sequencing, Geo-seq=geographical position sequencing, RCA= rolling circle amplification..
2
Major methods of spatially resolved transcriptomics
Method Principle Strengths Limitations
smFISH In situ hybridization via complementary base High sensitivity Low throughput
pairing Subcellular resolution
Quantitative
Without amplification bias
SRM Optical SRM and combinatorial labeling Improved throughput of smFISH Low throughput
ExFISH Physical magnification High-resolution imaging of RNA structure and Specific materials needed
location
seqFISH Sequential barcoding scheme Dramatically improved throughput Large number of designed probes needed
Optical crowding
Long workflow
MERFISH Error-robust encoding schemes achieved by Further improved throughput Large number of designed probes needed
introducing a certain Hamming distance Error detection and correction Optical crowding
Long workflow
corrFISH Cross-correlating the images from 2 of out of Decode high copy number RNAs Large number of designed probes needed
a total of 3 rounds of hybridization Long workflow
seqFISH+ Using secondary probes to dilute target Diluting the density of mRNA Large number of designed probes needed
transcripts Long workflow
tomo-seq Cryosectioning of tissue and performing RNA-
Genome-wide RNA tomography of tissues or Often results in 1D data along one axis
seq on individual thin sections organs Often only useful for elongated samples
Slide-seq DNA-barcoded beads surface Near-cellular spatial resolution No tissue imaging
Slide-seqV2 High throughput
sci-Space Labeling nuclei using unmodified DNA oligos Single-cell resolution Imaging restricted to nuclei
on glass slides Large capture area
Stereo-seq DNA nanoball (DNB) barcoded solid-phase Nano-level resolution (Subcellular) Imaging restricted to nuclei
RNA capture Large capture area
LCM-seq Applying laser energy to remove the cells of Speed, precision, and versatility Low throughput
interest under microscopy Suitable for a small number of cells Damages adjacent cells
RNA contamination
Geo-seq Combination of LCM and scRNA-seq Speed and precision Low throughput
Suitable for a small number of cells Damages adjacent cells
RNA contamination
NICHE-seq Cells sorted to scRNA-seq after Single-cell level RNA-seq Low throughput
fluorescent markers photoactivated by Reliant on transgenic mice
2-photon laser scanning microscopy Not suitable for clinical samples
ST Barcoded solid-phase RNA capture Combining histological staining Low spatial resolution
10X Visium Easy to perform Limited capture area
Requires no special instruments
Available commercial kit
HDST Barcoded solid-phase RNA capture Improved spatial resolution Limited capture area
DBiT-seq Microfluidic-based method to deliver Co-mapping of mRNAs and proteins Limited capture area
barcodes to the surface of a tissue slide Spatial resolution needs further
improvement
Seq-Scope Barcoded solid-phase RNA capture Nano-level resolution (Subcellular) Needs more research for demonstration
Large capture area
TIVA Photoactivatable mRNA capture in live cells Compatible with live and intact tissue, mRNA Low throughput
and tissues captured with precise spatial resolution,
with little bias from RNA contamination or
experimentally-related injury
FISSEQ Based on padlock probes and RCA, fixing the Preserve the tissue architecture for RNA Specific instruments required
cDNA fragments to the cellular protein matrix localization studies, localize subcellular RNA Random priming is inefficient and may
by amine cross-linker transcriptome-wide introduce bias
Large number of designed probes
needed
STARmap Based on padlock probes and RCA, 3D volumes of intact tissue, no intrinsic limit to Large number of designed probes needed
integrating hydrogel-tissue chemistry, the number of genes Workflow is complicated
targeted signal amplification, and In situ
SEDAL sequencing
Smart-3SEQ Combination of LCM and Smart-3SEQ Suitable for small samples or those whose RNA Needs more research for demonstration
is degraded
Visium FFPE Probes capture RNA after tissue For clinical samples from biobanks, Needs more research for demonstration
decrosslinked immunological staining can be performed
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  Chin Assoc of Blood Sci
the combination of imaging- and sequencing-based methods.
These methods have their intrinsic strengths and limitations,
with differences in target coverage, resolution, and throughput.
Like all methods, they must be appropriately matched to the
biological question at hand.
2.1. Imaging-based methods
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Spatial information about protein expression can be obtained
from traditional immunohistochemistry (IHC) approaches,
which are mediated by the specific binding reaction between
antigens and antibodies. In a variation of the IHC method,
spatial information about gene expression can be obtained by
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identifying the mRNA in cells using in situ hybridization (ISH),
which involves a hybridization reaction between labeled nucle-
otide probes and complementary targeting RNA molecules. The
single-molecule FISH (smFISH) method is the gold standard for
detecting transcripts, and efforts to develop multiplex FISH at
the transcriptome level are underway. This would allow FISH to
answer more complex biological questions.
2.1.1. smFISH: the gold standard for transcript detec-
tion smFISH combines quantitative FISH with digital imaging
microscopy to sensitively detect single RNA molecules using
probes. To amplify the signals, probes are often labeled with
multi-fluorochromes per molecule.9 However, the disadvantages,
which include difficulty in acquiring heavily labeled oligonucle-
otides, the easy loss of coupling fluorophores and self-quench-
ing, highly variable signals associated with multi-fluorophores
limit the system to a narrow range of applications. To overcome
these drawbacks, singly labeled probes, which are easier to gen-
erate and purify, were introduced to accurately detect individual
mRNA molecules with uniform signals10,11 (Fig. 1A).
smFISH can not only visualize the transcripts in isolated cells
or in situ tissues at a high resolution and with high hybridiza-
tion efficiency, but also provides information about the distri-
bution of subcellular RNA within cells. This feature means that
extra analyses concerning subcellular mRNA localization and
its functional stages can be achieved, as mRNA localization is
currently believed to contribute to the localized regulation of
gene expression.12–15 In addition, smFISH can precisely measure
the copy numbers of specific RNAs without amplification bias
and natural fluctuations in gene expression can be quantitatively
measured, permitting the elucidation of the mechanisms that reg-
ulate gene expression fluctuations and their role in a variety of
biological processes. These features make smFISH a robust tool,
and it is the standard method used for detecting transcripts.
2.1.2. Multiplexed FISH: improving target coverage Many
biological questions require transcription information about mul-
tiple genes, so efforts have been made to address the challenge
of the limited number of RNA species that be simultaneously
detected in cells using FISH. Super-resolution microscopy (SRM)
can resolve a large number of mRNAs in single cells by labeling
mRNAs with unique combinations of fluorophores. Although this
expanded the scalability of FISH from a few genes to about 30, it
was hard to make further progress due to the limited number of flu-
orophores.16 Later, a sequential barcoding scheme was introduced
to multiplex different mRNAs, called sequential barcoded FISH
(seqFISH).17 Here, the mRNA is barcoded by sequential rounds of
hybridization, imaging, and probe stripping. The number of RNA
species scales as FN, where F is the number of fluorophores and N
is the number of rounds of hybridization. Thus, the scalability of
seqFISH can be dramatically improved by increasing the number
of rounds of hybridization, despite the limited number of fluo-
rophores. In this manner, the entire human transcriptome can be
covered, including both mRNAs and non-coding RNAs (Fig. 1B).
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However, N rounds of hybridizations bring not only exponentially
increased throughput, but also exponentially increased detection
error rates. When N increases, the error rates increase rapidly, and
the majority of RNA molecules are likely to be misidentified after
16 rounds of hybridization.18 Therefore, multiplexed error-robust
FISH (MERFISH) method was designed. MERFISH is based on
combinatorial labeling and sequential imaging, and uses error-ro-
bust encoding schemes to correct labeling and detection errors.
The method introduces a certain Hamming distance to encode
RNAs, and the minimum Hamming distance is 4 (HD4 code),
meaning that at least 4 bits must be read incorrectly to change
one codeword into another. As a result, every single-bit error pro-
duces a word that is uniquely close to a single codeword, allowing
such errors to be detected and corrected. In addition, MERFISH
improves the multiplexity of transcripts that can be obtained in
single cells to 100 to 1000 through the use of binary sequential
barcodes19,20 (Fig. 1C).
2.1.3. Optical crowding: the major challenge for transcrip-
tome-level profiling Although seqFISH makes scaling to the
level of the genome theoretically possible, transcriptome-level
profiling in cells is hindered by optical crowding due to the
high density of global transcripts, even when combined with
SRM. Some progress has been made to improve the resolu-
tion of microscopy, with expansion microscopy (ExM) using
a swellable polyelectrolyte gel that can expand physically to
magnify the sample. RNAs are covalently attached to the gel,
a small molecule linker, and FISH (ExFISH) is performed in
cultured cells and intact tissues with high yield and specific-
ity.21 Another approach called correlation FISH (corrFISH) was
used in seqFISH to read out individual RNA species and bar-
codes. The principle is simple: RNA species are decoded such
that each RNA species appears in only 2 out of a total of 3
rounds of hybridization. By cross-correlating the images from
the 2 rounds of hybridization, only the specific RNA species will
generate a positive correlation. Although some algorithms have
been adapted due to the need for a high degree of temporal reso-
lution, corrFISH still represents a robust method to decode high
copy number RNAs in highly multiplexed seqFISH experiments,
using conventional fluorescence microscopy.22
Another way to address the issue of molecular crowding is
to dilute the density of the mRNA. Thus, seqFISH+ labels the
secondary probes with fluorophores rather than the primary
probes, which provide target sites for the secondary probes.
The primary probes divide the transcript set into 20 subsets,
and the secondary probes, which are labeled with three col-
ors, ultimately make it possible to visualize one-sixtieth of the
transcripts per image in each round of hybridization (Fig. 1D).
seqFISH+ achieves transcriptome-level profiling not only for
cultured cells but also in tissues, so that unbiased identification
of cells alongside their spatial organization can be realized.23
2.2. Sequencing-based methods
With the development of genetic techniques, next-generation
sequencing technology has become a powerful tool for tran-
scriptome analysis. scRNA-seq can precisely characterize cell
types and states at the molecular level, without bias, based on
global gene expression profiles.24 Generally speaking, sequenc-
ing-based methods are performed to capture or barcode the
RNA before reverse transcription to ensure each transcript can
be mapped to its original spatial spot, or are used to gain gene
expression information from cells in a certain spatial context.
RNA-seq can be conducted either ex situ or in situ.
2.2.1. Sequencing-based methods that do not validate
cell types Some methods do not provide the spatial resolu-
tion of microscopy-based techniques as they do not predict or
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Figure 1. Schematics of the main imaging-based methods. (A) In smFISH, fluorochrome-labeled probes detect individual mRNA molecules through in situ
hybridization. (B) In seqFISH, sequential barcoding: in each round of hybridization, a set of probes targeting different genes are hybridized on each transcript,
imaged, and then stripped with DNAse I. The same probe sequences are used in different rounds of hybridization, but probes are coupled to different fluoro-
phores. By decoding the temporal color sequences, the original genes can be identified. (C) In MERFISH, HD4 code is introduced by encoding probes contain-
ing a central RNA-targeting region flanked by 2 readout sequences. Encoding probes convert the RNA into a unique combination of readout sequences. (D) In
seqFISH+, I–IV sequences on the primary probes correspond to 4 rounds of barcoding, and the fourth round is used for error correction. Only one-twentieth
of the total genes in each fluorescent channel are labeled by readout probes in each hybridization readout round, lowering the density of transcripts in each
image. HD4 = minimum Hamming distance is 4, MERFISH = multiplexed error-robust FISH, seqFISH = seqFISH = sequential barcoded FISH, smFISH = sin-
gle-molecule FISH.

validate cell types using IHC or ISH. Examples of such methods
include tomo-seq and Slide-seq.25,26 Tomo-seq is a serial micro-
tomy-based sequencing method, which cryosections the tissue
of interest and performs RNA-seq on individual thin sections
(Fig. 2A). RNA extraction is performed for each individual
section and the RNA is barcoded with section-specific primers.
This approach can provide genome-wide RNA tomography of
tissues or organs, but it is often only possible to cryosection tis-
sue in 1 direction, resulting in 1-dimensional data along 1 axis
of the body. For this reason, tomo-seq is best suited for elon-
gated samples. Cells with complex organizations in the same
individual sections cannot be analyzed well, and often several
identical samples are required to perform cryosectioning in dif-
ferent directions along the main body axes to achieve 2- and
3-dimensional resolution.27
Unlike tomo-seq, Slide-seq is a method for transferring RNA
from tissue sections onto a surface covered in DNA-barcoded
beads with known positions, allowing the locations of the RNA

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  Chin Assoc of Blood Sci
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Figure 2. Schematics of the main sequencing-based methods. (A) In tomo-seq, specimens are cryosectioned, and sections are collected in individual micro-
tubes for spatially resolved transcriptomic analysis. (B) In LCM, an infrared laser melts the thermolabile polymer on a tissue section on a glass slide in the vicinity
of the laser pulse, resulting in the removal of polymer-cell composite from the tissue. A UV laser can cut away cells of interest or ablate unwanted tissue, leaving
cells of interest intact on the slide. (C) In NICHE-seq, tissue expressing a PA-GFP can be activated by 2-photon irradiation, allowing precise in situ labeling.
Activated cells are sorted to perform MARS-seq. (D) In 10X Genomics’ Visium, tissue sections are placed on a barcoded glass slide containing 4 capture areas,
each with around 5000 spatial spots. After HE staining and imaging, tissue permeabilization releases the mRNA and it is captured by spatial probes. LCM =
laser-capture microdissection, PA-GFP = photoactivatable green fluorescent protein, MARS-seq =massively parallel scRNA-seq, UV=ultraviolet, HE staining =
hematoxylin-eosin staining.

to be inferred by sequencing. It provides a scalable method for
obtaining spatially resolved gene expression data at resolutions
comparable to the sizes of individual cells.26 Slide-seqV2 is an opti-
mized version of Slide-seq with improvements in library genera-
tion, bead synthesis, and array indexing to reach an RNA capture
efficiency ~10-fold greater than Slide-seq.28 A new method, called
sci-Space, adapts the low-cost sci-Plex procedure for labeling or
“hashing” cell nuclei using unmodified DNA oligos to glass slides.
The hashed oligos spotted onto the glass slides are transferred
onto permeabilized nuclei (not intact cells) in freshly frozen tissue
sections. As the nuclei are stained with 4',6-diamidino-2-phenylin-
dole (DAPI) and the spatial coordinates are consequently imaged
during oligo transfer, sci-Space can retain a single-cell resolution.29
Obviously, the main limitation of all these methods is that the cell
types are purely identified by RNA-seq datasets, which lacks the
cell-validation process that microscopy-based techniques provide.

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 Duan et al
2.2.2. Sequencing-based methods that validate cell
types Although single-cell RNA-seq has the power to define
cell types or populations based on gene expression programs,
microscopy-based techniques such as IHC or ISH—which val-
idate candidate genes with high cell type specificity—remain
the gold standard to define cell type. Thus, a series of sequenc-
ing-based methods combined with imaging have been developed
to aid the study of solid cell types.
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Laser-capture microdissection (LCM) is an optional method
to obtain targeted cell subgroups or even single cells quickly
and precisely under the microscope. LCM directly harvests cells
of interest or isolates specific cells by cutting away unwanted
cells via infrared capture systems or ultraviolet cutting systems30
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(Fig. 2B). Histological staining or rapid antibody staining are
performed on the tissues before LCM so that information about
the types of cells captured is validated. The key advantages of
LCM are that the positional information of cells is maintained
and tissues are not dissected; in this way, subsequent analysis by
genomics, transcriptomics, or proteomics can be performed on
the LCM-captured cells.31–33 The extracted nucleic acids can be
amplified when the captured regions of interest are small, over-
coming the requirement for relatively large numbers of cells.
This advantage makes it possible to use LCM at the single-cell
level, meaning that it is especially useful for scarce tissues and
rare cell types. Geo-seq is the combination of LCM and scRNA-
seq technology.33 It is also practical to combine LCM with
Smart-seq2, a high-throughput scRNA-seq technology with
improved sensitivity, accuracy, and full-length coverage across
transcripts, making LCM plus Smart-seq2 a promising prospect
for SRT.31,34,35 However, the main obstacles to the wide appli-
cation of LCM SRT are its low throughput and potential RNA
contamination from other cells, as well as the inevitable damage
to adjacent tissue. This means that it is difficult to study the
interplay between individual cells and the microenvironment.
One method with similarities to LCM coupled with Smart-
seq2 is NICHE-seq, which couples with scRNA-seq. This tech-
nique combines photoactivatable fluorescent markers, 2-photon
laser scanning microscopy and flow cytometry-based fluores-
cence-activated cell sorting (FACS), and is coupled to massively
parallel scRNA-seq (Fig. 2C). NICHE-seq requires transgenic
mice that ubiquitously express a photoactivatable green fluo-
rescent protein (PA-GFP). Two-photon irradiation is used to
activate PA-GFP and in situ labeling. After tissue dissociation,
GFP-labeled cells can be sorted to perform scRNA-seq; thus,
this method combines information about the cell’s transcrip-
tional state and spatial information.36 As this method requires
transgenic mice expressing GFP, however, it is not suitable for
clinical samples.
Another method based on barcoded glass slides, called spa-
tial transcriptomics (ST), also validates cell types by histolog-
ical staining and imaging.37 RNA is spatially captured from
tissue sections and barcoded via a microarray on a glass slide.
Several primers with unique barcodes are placed on the micro-
scopic glass slide to create a microarray, and a section of tissue
is placed on top of the microarray surface. This tissue is then
fixed, then stained and imaged, before being permeabilized to
release RNA within the cells. In this way, transcripts from the
tissue are allowed to meet with the immobilized cDNA synthesis
primers in a reverse transcription reaction. Consequently, the
cDNA library is sequenced and the data are visualized together
with a high-resolution histological image of the tissue section.
Each slide consists of about 1000 spots, and ST is both easy to
perform and requires no special instruments. The method has
been acquired and optimized by 10x Genomics and a commer-
cial kit is available (Fig. 2D).
DBiT-seq is a microfluidic-based method that delivers bar-
codes to the surface of a tissue slide to allow for spatial omic
sequencing. This method enables the co-mapping of mRNAs
and proteins on a slide containing formaldehyde-fixed tissue38
(Fig. 3A). Immunofluorescence can be performed on the same
6
tissue slide, so that proteomics can be integrated with transcrip-
tomics. The capacity for integration is of great significance for
multi-omics research.
2.2.3. Sequencing-based methods for use in vivo Current
methods that capture RNA, such as LCM, cannot isolate mRNA
from individual in vivo cells without damaging adjacent tissue,
making it difficult to assess the influence of the microenviron-
ment on the transcriptome. To overcome this problem, a method
was engineered to extract mRNA from live cells or tissues at the
spatial resolution of a single cell. This method is called transcrip-
tome in vivo analysis (TIVA); it uses a photoactivatable bioti-
nylated tag, known as a TIVA-tag, that can penetrate the cell
membrane by virtue of a cell-penetrating peptide. Once inside
the cell, photoactivation is used to cleave the linkers between
the poly(U) sequence and two poly(A) stretches to enable the
poly(U) sequence to bind the poly-A tail of mRNAs (Fig. 3B).
With this method, mRNA can be noninvasively captured within
live cells and intact tissues, so it provides a useful tool to explore
the transcriptomes of single cells in the context of their natu-
ral microenvironment. However, the method is low throughput
with regard to the numbers of cells that can be analyzed.
2.2.4. Enhanced resolution and a larger area permit the
acquisition of more detail Solid RNA-barcoding is favor-
able in terms of its untargeted features and high coverage com-
pared with imaging-based methods. Efforts have been made to
improve the spatial resolution and capture area of this method,
as a higher resolution means the precise location of the tran-
scripts within the tissues, cells or even subcellular organelles can
be determined.
ST can achieve a resolution of only 100 μm (which is not at
the single-cell level), which means that the 100 μm spatial spots,
with a center-to-center distance of 200 μm, typically cover 5 to
100 cells each, depending on tissue type and region.37 Visium
technology from 10X Genomics has improved the ST method
by reducing the diameter of the capture area from 100 to 55 μm,
increasing the capture area to 42.25 mm2 (6.5 mm × 6.5 mm),
and decreasing workflow duration (Fig. 2D). Nevertheless, the
spatial resolution of 10X Genomics’ Visium is still insufficient to
resolve single cells in most samples.
Slide-seq and DBiT-seq both have improved spatial resolution
to 10 μm,26,38 but this is still a near-cellular resolution and cannot
be described as single-cell resolution. High-definition spatial tran-
scriptomics (HDST), an optimized version of ST, was designed
to improve ST resolution to the single-cell level. In this method,
hundreds of thousands of barcodes are randomly deposited onto
a slide at 2 μm resolution, and their positions are decoded by
sequential hybridization and error-correcting strategy.39 One
method, called spatiotemporal enhanced resolution omics-se-
quencing (Stereo-Seq), involves the deposition of a DNA nanoball
containing random barcoded sequences to form the chip. Each
spot is approximately 220 nm in diameter, with a center-to-cen-
ter distance of 500 nm.40 Another method, known as Seq-Scope,
has a center-to-center resolution of 0.5 to 0.8 μm (0.6 μm on
average).41 The nano-level distances of these methods are far less
than the diameter of single cells, giving them the high resolution
needed to determine the subcellular location of transcripts.
2.3. In situ sequencing methods
The combination of imaging and sequencing processes typ-
ically involves in situ sequencing methods. In situ sequencing
methods sequence cDNA amplicons based on padlock probes
and rolling circle amplification.42,43 The 2 ends of the padlock
probe can be ligated to form a circle-like structure upon hybrid-
ization, which then can be amplified using an isothermal DNA
polymerase. Amplification causes copies of the padlock probe
sequence to accumulate and form cDNA nanoballs. An amplified
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Figure 3. Schematics of DBiT-seq, TIVA, and STAR map. (A) In DBiT-seq, parallel microfluidic channels are used to deliver DNA barcodes to the surface of a
tissue slide and yield a 2D mosaic of tissue pixels. ADTs recognize a panel of proteins of interest, co-mapping mRNAs and proteins. (B) In TIVA, caged TIVA-
tags are loaded into cells or tissue by virtue of a disulfide-linked CPP. Selective photoactivation of the TIVA-tags in the desired cell or cells using a laser uncages
the TIVA-tags, and the exposed poly(U) can anneal to the poly-A tail of cellular mRNA. (C) In STAR map, only when both probes (SNAIL probes) hybridize to
the same RNA molecule can the padlock probe be circularized and rolling-circle-amplified to generate a DNA nanoball. The amplicons with an acrylic acid
N-hydroxysuccinimide moiety modification are copolymerized with acrylamide to embed within a hydrogel network. Gene identifiers are sequenced by in situ
SEDAL sequencing. ADTs = antibody-derived DNA tags, CPP = cell-penetrating peptide, STAR = spatially-resolved transcript amplicon readout mapping, TIVA=
transcriptome in vivo analysis, DBiT-seq =deterministic barcoding in tissue for spatial omics sequencing, SEDAL=sequencing with error-reduction by dynamic
annealing and ligation.

signal can be obtained by visualizing the cDNA nanoballs using FISSEQ, a highly multiplexed subcellular in situ RNA
fluorescently labeled detection oligonucleotides, which are com- sequencing method, uses random hexamer-primed reverse
plementary to the padlock probe. transcription in the presence of aminoallyl dUTP. Aminoallyl
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 Duan et al
dUTP then fixes the cDNA fragments to the cellular protein
matrix by a non-reversible amine cross-linker.44 The templates
are circularized after degrading the RNA, and subsequently
amplified using rolling circle amplification primers comple-
mentary to the adapter sequence introduced by RT. The ampl-
icons in the cells are then ready for sequencing-by-ligation
(SOLiD sequencing) at room temperature, and imaging on a
confocal microscope. To control the signal density of FISSEQ,
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a partition sequencing strategy was devised using pre-extended
sequencing primers.44,45 With 1 base extended, one can ran-
domly sample amplicons at 1/4th of the original density, and
with 2 bases extended sampling at 1/16th of the density is pos-
sible. One notable highlight of FISSEQ is that it preserves the
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tissue architecture for RNA localization studies, and localizes
subcellular RNA transcriptome-wide. However, the random
priming component is inefficient and may introduce bias via
uneven binding efficiency, which is due to differences in base
composition.
While SRT can be performed on tissues or sections on a slide,
applying in situ sequencing methods to 3-dimensional volumes
of intact tissue is not easy. To achieve this goal, a method known
as spatially resolved transcript amplicon readout mapping
(STARmap) integrates hydrogel-tissue chemistry, targeted signal
amplification and in situ sequencing.46 The method bypasses the
reverse transcription step and uses the SNAIL approach instead,
which achieves specific amplification of nucleic acids via intra-
molecular ligation using a pair of custom primer and padlock
probes. Only when both probes hybridize to the same RNA
molecule can the padlock probe be circularized and rolling-cir-
cle-amplified to generate a DNA nanoball. The cDNA ampli-
cons are modified and copolymerized in situ to embed within a
hydrogel network. In situ SEDAL sequencing was devised specif-
ically for STARmap, to sequence 5-base barcoded gene-specific
identifier segments in SNAIL probes over 6 rounds of hybridiza-
tion (Fig. 3C). The highlight of STARmap lies in its ability to be
used in 3-dimensional in situ transcriptomics, which is undoubt-
edly a state-of-the-art SRT technique and serves to deepen our
understanding of anatomy and transcriptomics. In parallel,
there is no intrinsic limit to the number of genes or RNA spe-
cies that can be simultaneously and quantitatively accessed with
STARmap, since it can be adapted to longer sequencing lengths
or higher gene numbers.
In conclusion, ISH is highly sensitive, and can detect tran-
scripts at a subcellular resolution. Many powerful strategies
have been designed to improve the target coverage of ISH
to the transcriptome level and tackle the problem of opti-
cal crowding. However, such methods are typically limited
by long image acquisition times and a complex probe-de-
signing process. While next-generation sequencing technol-
ogy enables high-throughput transcriptomic profiling, many
sequencing-based methods are much easier to perform once
suitable experimental platforms have been established. As
spatially barcoded beads or spots can be arrayed more and
more densely, some solid-barcoded methods can reach a cellu-
lar or subcellular resolution, and spatial information is more
precisely retained. To some extent, in situ sequencing methods
are a combination of imaging- and sequencing-based methods,
sequencing gene identifiers in the designed primers via FISH.
Although in situ sequencing methods are high throughput, the
complexity of designing targeted probes cannot be ignored
(Table 1).
Most of the in situ-based methods described above rely on the
use of fresh tissues. However, fresh clinical material is often dif-
ficult to obtain for research purposes. To remove the barriers to
clinical research, some methods have been designed to use for-
malin-fixed paraffin-embedded (FFPE) tissue, in which the RNA
is degraded. Smart-3SEQ enables large gene expression profiling
experiments to be conducted on even small amounts of total
RNA, and effectively characterizes small samples extracted by
LCM from FFPE tissue.47 In addition, 10X Genomics markets a
8
commercially available system, called Visium FFPE, that enables
SRT to be performed on samples from a biobank. Some stud-
ies have already successfully used 10x Visium FFPE on FFPE
tissue.48
3. DATA ANALYSIS IN SRT
The additional dimension of spatial information brings not
only a novel perspective on the transcriptome, but also signif-
icant challenges for data analysis due to increased data vol-
ume and complexity. Generally speaking, strategies for SRT
data analysis include computational approaches specifically
designed for SRT and integration methods (with bulk or sin-
gle-cell RNA-seq data). Different analytical approaches have
been developed for localized gene expression pattern identifi-
cation, spatial decomposition, gene imputation, and cell–cell
communication.49
Gene expression and spatial location are both of great
relevance to biological functions. By analyzing the relation-
ship between gene expression and spatial location, some
approaches, including Trendsceek,50 SpatialDE,51 SPARK,52
SPARK-X,53 sepal,54 SpaGCN,55 and GLISS, are designed to
identify localized gene expression patterns and spatially vari-
able genes (SVGs) based on different algorithms. Another way
to discover SVGs is spatial clustering. Unlike standard clus-
tering methods for scRNA-seq, spatial clustering is for the
capture locations. Methods like stLearn, MULTILAYER,56
BayesSpace,57 SC-MEB,58 and STAGATE can be applied for
spatial clustering. As most of the sequencing-based methods
do not guarantee that a single capture spot contains RNA
from only 1 cell, a capture location is often a mixture of mul-
tiple cell types. Thus, some spatial decomposition algorithms,
such as NNLS, spatialDWLS,59 SPOTlight,60 RCTD,61 DSTG,
Tangram, and Cell2location attempt to infer the proportions
of cell types in each spot, or score a spot for how strongly
it corresponds to a single cellular subtype based on bulk or
single-cell RNA-seq data. However, certain SRT methods,
particularly those that are imaging-based, cannot provide
deep transcriptomic information due to limited gene cover-
age. Gene imputation provides a chance to impute the missing
genes from scRNA-seq data in order to improve the quality
of SRT data. Methods that include gene imputation functions
include Tangram,62 gimVI, Harmony,63 LIGER,64 Seurat,65
SpaGE,66 and stPlus.67 In addition, the reconstruction of spa-
tial information for scRNA-seq data can be achieved by map-
ping, which leverages a small set of landmark genes to map
the single cell back to its context. Methods that incorporate a
mapping function include Harmony, LIGER, Seurat, SpaGE,
DEEPsc,68 DistMap, SpaOTsc,69 novoSpaRc,70 and CSOmap.71
In addition, cell–cell interactions can be analyzed using some
standard algorithms, based on the ligand–receptor interaction
pairs from scRNA-seq data and a database of known ligand–
receptor interactions. However, the loss of spatial information
in scRNA-seq data may lead to inaccurate detection of ligand–
receptor interactions, as cellular cross-talk is often spatially
restricted. Therefore, SRT data might potentially evaluate the
reliability of the ligand–receptor interactions computed from
scRNA-seq. In this manner, integrating scRNA-seq with SRT
data may accurately reveal specific cell subpopulations and
their interactions. Approaches to study cell–cell interactions
include SVCA,72 GCNG, NCEM, MISTy, stLearn, Squidpy,
novoSpaRc, SpaOTsc, and DEEPsc. Among these meth-
ods, comprehensive benchmark studies are needed to help
users select methods that best fit their data and hypotheses.
According to a study benchmarking 16 integration methods,
Tangram, gimVI, and SpaGE outperformed other integration
methods when predicting the spatial distribution of RNA
transcripts, while Cell2location,73 SpatialDWLS, and RCTD
were the top-performing methods for the cell type deconvo-
lution of spots.74
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  Chin Assoc of Blood Sci
In conclusion, SRT and scRNA-seq data complement each
other, since SRT methods cannot always maintain single-cell res-
olution with the same depth and whole transcriptome coverage
of scRNA-seq. The integration of scRNA-seq and SRT data could
improve cell type annotation, cell clustering, spatial decompo-
sition, gene imputation, and spatial location reconstruction. A
scRNA-seq database could form a solid foundation for SRT data
analysis. However, the proper handling of spatial information
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obtained from SRT data remains a challenge in terms of data
processing, and the establishment of proper protocols will be an
essential step to reach the full potential of these methods.
4. APPLICATIONS FOR SRT
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At present, SRT technologies are widely utilized in basic and
clinical research, especially in neuroscience, cancer biology, and
developmental biology. However, there are likely further appli-
cations for SRT that are yet to be fully utilized, including in the
study of hematopoeisis. We reviewed the potential applications
in this field in Figure 4.
4.1. Neuroscience: unraveling the organization of brain
cell types and functions
The diversity of brain cell types and the complex organization
of the brain have long been major challenges to our understand-
ing of brain functions during physiological and pathological
conditions.75 A census of brain cell types and their functions will
pave the way for systematic research and, potentially, even novel
clinical treatments. SRT has advantages over scRNA-seq due to
the retained anatomical and spatial contexts, and various spa-
tially resolved transcriptomic methods have been applied to map
cell types in neuroscience. For example, an atlas of the mouse
somatosensory cortex was built using osmFISH, a semiautomated
version of smFISH. Based on scRNA-seq data, the study identified
31 clusters of cell types and 33 cell type-specific marker genes.76
Although multiplexing in osmFISH is relatively low, its sensitivity
is higher than scRNA-seq and can provide single-cell-level spatial
resolution for gene detection. Multiplexed FISH techniques, such
as MERFISH and seqFISH, are applied when more genes need
to be analyzed. The multiplexing of 250 genes using seqFISH
identified distinct subregions of the hippocampus that contained
different combinations of cell types.18 In addition, MERFISH
combined with measurements of immediate early gene expression
identified ~70 neuronal populations in the mouse hypothalamic
preoptic area, and defined discrete cell populations activated by
specific social behaviors.77 STARmap has also been used to recon-
struct the 3D locations of cells in the murine primary visual cor-
tex and medial prefrontal cortex; in this particular study, >1000
genes were simultaneously mapped in mouse brain sections at a
single-cell resolution.46 Sequencing-based methods such as Slide-
seq and 10X Genomics’ Visium have also been used to analyze
the global transcriptome in both the mouse and human brain.26,37
The application of spatially resolved transcriptomic methods
is highly likely to improve our understanding of neurological
diseases. For example, SRT has already revealed the spatiotem-
poral dynamics of molecular pathology in amyotrophic lateral
sclerosis.78 Furthermore, in a murine model of Alzheimer dis-
ease, ST and in situ sequencing were used to better characterize
the dysregulated cellular network in the early and late phases of
disease. The results of this study provided profound insights into
the pathogenesis of Alzheimer disease, and also provided clues
concerning potential therapeutic targets.79
4.2. Cancer: profiling the spatial heterogeneity of tumors
and the tumor microenvironment
Heterogeneity both within and between tumors is found in
most types of cancer.80,81 The expansion of tumor subclones
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with varying genetic alterations and the interactions between
tumor cells and the tumor microenvironment presents a compli-
cated situation that influences disease development.82 Profiling
the spatial heterogeneity of tumors and the tumor microen-
vironment will provide insights into tumor progression and
responses to treatment, and will therefore assist in the diag-
nosis and treatment of this condition. ST technology has been
used to identify intertumoral and intratumoral heterogeneity in
multiple types of cancer. Furthermore, changes in gene expres-
sion during prostate cancer and cutaneous malignant mela-
noma progression have revealed a detailed landscape of tumor
progression and metastases.83,84 Several features of the tumor
microenvironment in primary liver cancer, including stromal
and immune cell distribution, tumor cluster interaction, can-
cer stem cell-niche diversity and tertiary lymphoid structure
composition have been characterized.85 Coupling SRT with
other technologies also improves its capacity to characterize
tumors: for example, when SRT was combined with scRNA-
seq, tissue architecture and interactions between cell subpopu-
lations were revealed in pancreatic ductal adenocarcinomas.86
A tumor-specific keratinocyte population, immune infiltrates,
and heterogeneity at tumor leading edges have also been iden-
tified in human squamous cell carcinoma using multimodal
analysis, which included scRNA-seq and ST.87 The heteroge-
neity of breast cancer has also been characterized, and several
diagnostic and prognostic markers proposed.88,89 Additionally,
by integrating spatial gene expression and HE-stained images
of breast tumor, a deep learning algorithm was developed to
predict the spatially resolved transcriptome of a tissue from
the images, enabling image-based screening for molecular bio-
markers.90 To guide clinical treatment, biomarkers associated
with beneficial PD-1 checkpoint blockade in non-small-cell
lung cancer were identified using digital spatial profiling.91
The ST analysis of human bladder cancer also identified an
N-Cadherin 2-expressing epithelial cell subpopulation which
could be used to predict therapeutic response, which could
guide treatment decisions.92 In conclusion, these findings pro-
vide novel insights into the complex ecosystem of cancer. and
have the potential to improve individualized cancer treatments
and drug discovery.
4.3. Developmental biology: characterizing the
temporal and spatial expression blueprint of embryonic
development
Embryonic development is a complex process where dynamic
changes rapidly occur at the biomolecular level.93,94 Lineage
tracing and fate mapping are powerful tools in developmen-
tal biology, providing a picture of the lineage hierarchy and
linking the initial cell position to future fate.95 With the emer-
gence of SRT, analysis of both the temporal and spatial aspects
of embryonic development can be performed, and a temporal
and spatial expression blueprint is eagerly anticipated. In the
early embryo, gastrulation is an essential developmental event,
leading to the formation of gastruloids, which are aggregates
of embryonic stem cells.96,97 Applying Geo-seq to the germ lay-
ers from pre- to late-stage gastrulation enabled the molecular
genealogy of tissue lineages and the continuum of pluripotency
states in time and space to be revealed. Furthermore, the molec-
ular determinants that drive lineage specification and tissue
patterning were identified.98 Tomo-seq and scRNA-seq revealed
genome-wide gene expression patterns in mouse gastruloids and
embryos, and identified various embryonic cell types that were
not previously known to be present in gastruloids. Based on
their results, the study’s authors proposed that somitogenesis
occurs in gastruloids.99 In addition, ISH has determined the gene
expression pattern in gastruloids generated from embryonic
stem cells, to validate an in vitro model of early anteroposterior
organization during human development.100 Stereo-seq has also
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Figure 4. Applications for SRT. In neuroscience, SRT was used to unravel the organization of brain cell types and functions. In cancer biology, the spatial het-
erogeneity of tumors and the tumor microenvironment can be characterized. In developmental biology, SRT was used to characterize the temporal and spatial
expression blueprint of embryonic development. In hematology, most studies utilizing SRT concern embryonic hematopoiesis, and aim to clarify the distinct
waves of developmental hematopoiesis. AGM = aorta-gonad-mesonephros, HSCs = hematopoietic stem cells, SRT = spatially resolved transcriptomics.

reconstructed the spatially resolved developmental trajectories
of cell-fate transitions and molecular changes during zebrafish
embryogenesis.101
Some research has also focused on the formation of organs in
embryonic development. The large field-of-view and cellular res-
olution of Stereo-seq has been used to map the spatiotemporal
transcriptomic dynamics during mouse organogenesis.102 ST was
also used to study cardiac morphogenesis, and the spatiotempo-
ral organ-wide gene expression and cell atlas of the developing
human heart was established during 3 developmental stages.103
Morphogenesis of the human intestine across time, location,
and cellular compartments has been charted by both scRNA-
seq and 10X Genomics’ Visum.104 The gene expression profile of
fetal livers from 8 to 17 weeks post-conception in humans has
also been illustrated using 10X Genomics’ Visium.105
4.4. Hematopoiesis: shedding light on the distinct
waves of developmental hematopoiesis, and making
breakthroughs in adult bone marrow
Hematopoiesis is the process by which blood cells are con-
tinually replenished throughout the lifetime of an organism,
by virtue of the self-renewal and differentiation of hematopoi-
etic stem cells (HSCs) and the regulation of the local micro-
environment, or niche.106 Distinct waves of hematopoiesis have
been defined during embryogenesis, including both primitive
10
and definitive hematopoiesis.107,108 SRT technologies have been
applied in developmental hematopoiesis to explore the genera-
tion and expansion of HSCs, as well as the regulation of their
niche. Geo-seq, combined with bulk and single-cell RNA-seq,
was used to examine the caudal hematopoietic tissue of zebraf-
ish (the counterpart of the mammalian fetal liver) and a detailed
spatiotemporal transcriptome of hematopoietic stem and pro-
genitor cells (HSPCs) and niche during HSPCs expansion was
characterized.109 Meanwhile, LCM-seq has been used in the
human embryonic aorta-gonad-mesonephros (AGM) region to
identify secretory factors that promote human HSC develop-
ment.110 Another study combined with 10X Genomic’s Visium
and Stereo-seq identified novel HSC/MPP pocket-like units
(HSC PLUS) composed of niche cells and enriched with growth
factors; providing an essential resource for understanding HSC/
MPP development in the fetal liver.111 In addition, 10X Genomic’s
Visium was used to visualize HSC emergence in human embry-
onic tissues, including the liver, AGM, gut, and vitelline and
umbilical vessels, validating the generation of AGM-like defini-
tive HSPCs from human pluripotent stem cells.112
scRNA-seq technologies have been widely performed in the
adult hematopoietic system, and have confirmed the heteroge-
neity of HSPCs.113 However, SRT is yet to be applied in this
field. Profiling the SRT of adult bone marrow would be of great
significance because this will provide an opportunity to study
the interplay between HSPCs and their niche under both nor-
mal conditions and stress. Most SRT methods require an intact
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  Chin Assoc of Blood Sci
slice of tissue, which may be challenging for adult bone marrow
due to the presence of calcified bone. Therefore, tissue prepa-
ration and cryosectioning need to be optimized. Furthermore,
bone marrow is composed of various hematopoietic cells and
non-hematopoietic cells, including mesenchymal stromal cells,
pericytes, fibroblasts, and endothelial cells.114,115 Importantly,
bone marrow hematopoietic cells are usually smaller than other
cell types, and the cells are usually distributed evenly in the bone
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marrow, lacking any apparent structure in terms of anatomi-
cal or functional division.116,117 These intrinsic features of bone
marrow impede the experimental applications of SRT, as well
as associated data processing. LCM-seq has been applied to
study adult bone marrow to reveal bone marrow niche organi-
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zation, setting an excellent example for future research.118 RNA-
barcoding methods with improved resolution, such as Slide-seq,
HDST and stereo-seq, are recommended for bone marrow, since
the cellular or subcellular resolution is the key to compensating
for smaller cells and diverse cell types. Furthermore, different
types of HSPCs share high similarities in transcriptomics. Thus,
we propose that SRT data can be properly analyzed only when
combined with scRNA-seq data. Of note, in the past decade,
single-cell transcriptomic data of bone marrow hematopoietic
cells and niche cells has been collected, serving as a supporting
resource for SRT.114,115
5. CONCLUSIONS AND FUTURE PERSPECTIVES
SRT technologies provide an opportunity to uncover the
molecular architecture of tissues. Methods that are more scal-
able, have a higher resolution and easier workflows are expected
to be developed, and more results produced by SRT should be
published soon. For the remainder of this review, we anticipate
future developments that will unlock the full potential of SRT in
both biological research and the clinic.
5.1. Realizing single-cell SRT
The correct association of detected mRNAs and single cells is
a great challenge for both imaging- and sequencing-based meth-
ods. For most sequencing-based methods, such as Slide-seq and
ST,26,35,37 the spatial information is retained in the form of dif-
ferentially barcoded spots. Although some methods, including
HDST, Stereo-seq and Seq-Scope, have achieved cellular or sub-
cellular resolution,39–41 the spot boundaries do not correspond
to the cell boundaries, meaning that transcripts captured in the
same spot do not always come from a single cell. Furthermore,
it cannot be guaranteed that the 10 μm-thin slices usually
obtained during tissue sampling will contain a single layer of
cells. This means that the transcripts of all the cells on the Z-axis
or the vertical axis of the slide will be captured on the same
spot. In imaging-based methods, the lack of cell boundaries
makes it hard to associate individual mRNA molecules with the
correct cells. In such methods, DAPI-stained nuclei may be the
only method to determine cell outlines, especially when differ-
ent cell types are very close together. In Sci-Space, which lever-
ages sci-Plex to transfer DNA oligos into permeabilized nuclei,
DAPI-stained nuclei and spatial coordinates of oligo transfer are
co-registered. The nuclei from the tissue on the slide are also
extracted to enable sci-RNA-seq.29 Although Sci-Space retains
a single-cell resolution, it is hard to avoid contamination from
adjacent cells. Moreover, the nuclei-assembling strategy may
collapse when applied to tissues that contain irregularly shaped
cells, such as neurons and glial cells in the brain. Regardless,
we expect that more robust approaches will ultimately permit
the characterization of the whole transcriptome of single cells.
The elaboration of some cytomembrane-outlining strategies,
such as immunofluorescent staining or auto-fluorescent reporter
animals to visualize the plasma membrane, may help define cell
boundaries and realize the potential of single-cell SRT. Existing
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rapid membrane imaging methods, such as fluorescent probe-
based methods, have already been used to facilitate in situ
plasma membrane imaging of neurons and erythrocytes in the
complex environment of the brain.119 However, optimization of
in situ plasma membrane imaging in combination with SRT still
requires further work.
5.2. Integrating spatially resolved multi-omics to advance
our understanding of biological systems
SRT is far more than just transcriptomics; rather, the tech-
nique is empowered by the retained positional information
provided by in situ methods. The locations of cells and the
microenvironment play an important role in uncovering cell-to-
cell and cell-to-extracellular matrix interactions, and the inte-
gration of functional and spatial information is of great benefit
in biological studies. Spatially resolved transcriptomic methods
provide several invaluable methods that can also be adapted to
suit certain other in situ omics. We expect that SRT will inspire
the development of other spatially resolved omics beyond
transcriptomics, such as in situ epigenomics, epitranscriptom-
ics, proteomics, and metabolomics, which will help us better
understand different aspects of cell function. Epigenomics is a
reliable tool to map chromatin accessibility dynamics and high-
er-order chromatin structure, and adding spatial information to
this would surely enable new levels of understanding of cell-
fate decisions, identity, and function.120,121 Epitranscriptomics
concerns a wide range of post-transcriptional RNA modifi-
cations, and spatial epitranscriptomics enables studies under
specific microenvironments, broadening our understanding of
the spatial regulation of gene expression.122 In parallel, metab-
olomics transcends genomics and proteomics, representing the
most downstream metabolic stage. The metabolome is widely
accepted to constitute a dynamic and sensitive measure of phe-
notype, placing metabolomics at the forefront of biomarker
discovery.123 Several spatially resolved metabolomic technolo-
gies have already been applied in several fields, such as tumor
metabolism, allowing metabolites and metabolic enzymes to be
directly discovered and studied in their native state.124–127
On the other hand, cell states that are defined based on data
from one type of omics may not be accurate and comprehensive,
and cell states defined by different omics may not always be in
accordance. We therefore expect the emergence of more spatially
resolved multimodal technologies, both at the experimental
level and in computational analysis, to be able to simultane-
ously profile multiple data types in the same cell. For exam-
ple, DBiT-seq can capture both proteins and transcripts on the
same sequencing slide, so that proteomics and transcriptomics
can be simultaneously performed on the same cells or tissues.38
We are also appreciative of and enthusiastic about methods that
validate cell types, not just to predict transcriptomic states, but
more importantly to provide more possibilities for multi-omic
research. For example, some sequencing-based methods with
high spatial resolution, such as Stereo-seq, involve the rapid
staining and imaging of just the tissue nuclei to avoid mRNA
degradation.40 Simultaneously retaining the mRNA, protein or
metabolites in the same cells may be very challenging. Proper
tissue preparation and fixation are essential to preserve the tis-
sue, and so existing workflows containing these steps will need
to be optimized to suit multimodal research.
ACKNOWLEDGMENTS
This work was supported by grants from the Ministry of
Science and Technology of China (2021YFA1100900 and
2020YFE0203000), the National Natural Science Foundation of
China (81730006, 81922002, 81861148029, and 81870086),
CAMS Innovation Fund for Medical Sciences (2021-I2M-1-040
and 2021-I2M-1-019), Haihe Laboratory of Cell Ecosystem
11
 Duan et al
Innovation Fund (HH22KYZX0016) and Distinguished Young
Scholars of Tianjin (19JCJQJC63400).
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