Integration of Computational Analysis and Spatial Transcriptomics in Single-cell Studies

Recent advances of single-cell transcriptomics technologies and allied computational methodologies have revolutionized molecular cell biology.Meanwhile,pioneering explorations in spatial transcriptomics have opened up avenues to address fundamental biological questions in health and diseases.Here,we review the technical attributes of single-cell RNA sequencing and spatial transcriptomics,and the core concepts of computational data analysis.We further highlight the challenges in the application of data integration methodologies and the interpretation of the biological context of the findings.

Comments on ‘Molecular architecture of lineage allocation and tissue organization in early mouse embryo’

Single-cell RNA-seq,with its capability to align cells of continuously changed status by pseudo-time reconstruction,has greatly revolutionized the understanding of cell fate transition during embryo development(Shapiroetal.,2013;Hoppeetal.,2014).

The genome-wide molecular regulation of mouse gastrulation embryo

The diverse morphologies among vertebrate species stems from the evolution of a basic body plan that is constituted by a spatially organized ensemble of tissue lineage progenitors. At gastrulation, this body plan is established through a coordinated morphogenetic process and the delineation of tissue lineages that are driven by the activity of the genome. To explore the molecular mechanisms, in a comprehensive context, it is imperative to glean an understanding of the region-and population-specific genetic activity underpinning this fundamental developmental process. In this review, we outline the recent progress and the future directions in studies of genome activity for the regulation of mouse embryogenesis at gastrulation.

Connecting past and present:single-cell lineage tracing

Central to the core principle of cell theory,depicting cells'history,state and fate is a fundamental goal in modern biology.By leveraging clonal analysis and sin-gle-cell RNA-seq technologies,single-cell lineage trac-ing provides new opportunities to interrogate both cell states and lineage histories.During the past few years,many strategies to achieve lineage tracing at single-cell resolution have been developed,and three of them(in-tegration barcodes,polylox barcodes,and CRISPR barcodes)are noteworthy as they are amenable in experimentally tractable systems.Although the above strategies have been demonstrated in animal develop-ment and stem cell research,much care and effort are still required to implement these methods.Here we review the development of single-cell lineage tracing,major characteristics of the cell barcoding strategies,applications,as well as technical considerations and limitations,providing a guide to choose or improve the single-cell barcoding lineage tracing.

Ectodermal progenitors derived from epiblast stem cells by inhibition of Nodal signaling

The ectoderm has the capability to generate epidermis and neuroectoderm and plays imperative roles during the early embryonic development.Our recent study uncovered a region with ectodermal progenitor potential in mouse embryo at embryonic day 7.0 and revealed that Nodal inhibition is essential for its formation.Here,we demonstrate that through brief inhibition of Nodal signaling in vitro,mouse embryonic stem cell(ESC)-derived epiblast stem cells(ESD-EpiSCs)could be committed to transient ectodermal progenitor populations,which possess the ability to give rise to neural or epidermal ectoderm in the absence or presence of BMP4,respectively.Mechanistic studies reveal that BMP4 recruits distinct transcriptional targets in ESD-EpiSCs and ectoderm-like cells.Furthermore,FGF–Erk signaling may also be alleviated during the generation of ectoderm-like cells.Thus,our data suggest that instructive interactions among several extracellular signals participate in the commitment of ectoderm from ESD-EpiSCs,which shed new light on the understanding of the formation of ectoderm during the gastrulation in early mouse embryo development.

Single-cell technologies:From research to application

In recent years,more and more single-cell technologies have been developed.A vast amount of single-cell omics data has been generated by large projects,such as the Human Cell Atlas,the Mouse Cell Atlas,the Mouse RNA Atlas,the Mouse ATAC Atlas,and the Plant Cell Atlas.Based on these single-cell big data,thousands of bioinformatics algorithms for quality control,clustering,cell-type annotation,developmental inference,cell-cell transition,cell-cell interaction,and spatial analysis are developed.With powerful experimental single-cell technology and state-of-the-art big data analysis methods based on artificial intelligence,the molecular landscape at the single-cell level can be revealed.

Lung development and regeneration:newly defined cell types and progenitor status

The lung is the most critical organ of the respiratory system supporting gas exchange.Constant interaction with the external environment makes the lung vulnerable to injury.Thus,a deeper understanding of cellular and molecular processes underlying lung development programs and evaluation of progenitor status within the lung is an essential part of lung regenerative medicine.In this review,we aim to discuss the current understanding of lung development process and regenerative capability.We highlight the advances brought by multi-omics approaches,single-cell transcriptome,in particular,that can help us further dissect the cellular player and molecular signaling underlying those processes.

Generation of functional posterior spinal motor neurons from hPSCs-derived human spinal cord neural progenitor cells

Spinal motor neurons deficiency results in a series of devastating disorders such as amyotrophic lateral sclerosis(ALS),spinal muscular atrophy(SMA)and spinal cord injury(SCI).These disorders are currently incurable,while human pluripotent stem cells(hPSCs)-derived spinal motor neurons are promising but suffered from inappropriate regional identity and functional immaturity for the study and treatment of posterior spinal cord related injuries.In this study,we have established human spinal cord neural progenitor cells(hSCNPCs)via hPSCs differentiated neuromesodermal progenitors(NMPs)and demonstrated the hSCNPCs can be continuously expanded up to 40 passages.hSCNPCs can be rapidly differentiated into posterior spinal motor neurons with high efficiency.The functional maturity has been examined in detail.Moreover,a co-culture scheme which is compatible for both neural and muscular differentiation is developed to mimic the neuromuscular junction(NMJ)formation in vitro.Together,these studies highlight the potential avenues for generating clinically relevant spinal motor neurons and modeling neuromuscular diseases through our defined hSCNPCs.