Human Developmental Cell Atlas: milestones achieved and the roadmap ahead

59 The Human Developmental Cell Atlas (HDCA), as part of the Human Cell Atlas, aims to generate 60 a comprehensive reference map of cells during development. This detailed study of development 61 will be critical for understanding normal organogenesis, the impact of mutations, environmental 62 factors and infectious agents on congenital and childhood disorders, and the molecular cellular 63 basis of ageing, cancer and regenerative medicine. In this perspective, we outline the challenges 64 of mapping and modelling human development using state of the art technologies to create a 65 reference atlas across gestation for scientific and clinical benefit. We discuss the potential value 66 of HDCA to enhance human pluripotent stem cell-derived organoid model systems and, in turn, 67 the use of organoids and animal models to inform HDCA. Finally, we provide a roadmap towards 68 a complete atlas of human development. biological developmental biology, embryology, genetics, model systems; clinical specialties: maternal/fetal health, pediatrics, in vitro fertilization, clinical genetics, histology;


Introduction
Historically, most modern developmental biology research focused, by necessity, on model 71 organisms. Due to practical challenges, human development, from a fertilized ovum to a fully 72 formed fetus at birth, has remained a poorly understood 'black box'. The implications for 73 understanding human development are far-reaching, as many congenital disorders and childhood 74 cancers may originate during susceptible windows of development [1][2][3][4] . The clinical relevance 75 extends into adulthood for ageing, cancer and applications in regenerative medicine and stem cell 76 therapy 5,6,7 . Furthermore, embryonic and fetal stem cells and developmental trajectories provide 77 an essential reference and guide for engineering pluripotent stem cell (PSC)-derived organoids 8 .

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For these reasons, a cell atlas of human development will have far-reaching impacts that enhance 79 developmental biology research based on model organisms. Early studies of human embryogenesis began through morphometric and qualitative assessments 82 of human embryos (Figure 1). The Carnegie staging system, a valuable resource that is still widely 83 used, is one example that emerged from these pioneering studies 9 . Advances in imaging, 84 cytometry and genomics technologies revealed further insights into the complex four-dimensional 85 spatio-temporal changes and cellular architecture during organogenesis 10 . Recent progress in single cell profiling technologies has revolutionised our ability to study human 93 development at unprecedented resolution 11 . Computational methods for single cell genomics data 94 collected from multiple organs and developmental stages have enabled us to define the wealth of 95 developmental cell states and infer developmental trajectories of transitional populations between 96 them. Although data is collected from serial static snapshots across development, because the 97 process is asynchronous, computational algorithms can infer both continuous temporal 98 progressions and the underlying regulatory programs driving them 12,13 . Emerging spatial profiling 99 methods now allow us to map the temporal progression in 2-and 3D spatial context 14 . What is a developmental cell atlas? 110 Reminiscent of the Greek god Atlas, the developmental cell atlas will hold measurements and 111 information about the cells of the developing human, from the earliest stage through fetal life up 112 to birth, spanning multiple modalities that can be used as reference and for interrogation to derive 113 new understanding. From these measurements, the atlas will abstract the census of cells    The challenges posed by the temporal nature of the developmental atlas are also its key strength 152 as dynamics can be powerfully harnessed to elucidate the regulatory mechanisms underlying these 153 processes. Understanding the mechanisms that endow developing cells with their plasticity can be 154 employed to improve regenerative therapies and will provide insight into how cancer cells exploit     The field of developmental biology has traditionally drawn on ontogenic relationships to define 257 cell types, but this is challenging in humans, where information is often captured as a snapshot    of morphogenesis and pattern formation in development 61 . It is likely that many additional 346 emergent properties of cells and their ecosystems will be discovered using such an interdisciplinary 347 approach and some of these will need new vocabularies, ontologies and modeling approaches to 348 uncover and understand. These approaches will need to consider vast and computable multi-omics 349 data, concurrently model state, position, internal and external factors and environment, and be able 350 to predict the state and 3D location of many components across time as development unfolds.   Organ atlases of lung, heart, gastrointestinal tract, kidney, germ cells and gonads, and brain (Table     is not observed in humans; and by contrast, KLF17 is enriched in the human, but not mouse 96 .

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FGF8, required for mouse gastrulation, is not needed in the human embryo of the same stage 97 .

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Due to the availability of in vivo experimental systems to study lineage mapping in animal models, 443 comparative biology has the potential to make major contributions to one of the most pervasive 444 issues of single cell biology, namely cell ontology, including its relevance, utility and limitations.

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Comparative lineage reconstruction also represents one of the more promising approaches to 446 connect human developmental datasets with those key early stages of mammalian development 447 that are largely inaccessible for human studies, such as the first few weeks after implantation.   The initial HCA White paper emphasized 12 distinct organ systems within the human body, and 518 highlighted the importance of a developmental cell atlas. Integrated multi-organ analyses will 519 provide novel insights on tissue microenvironment shaping resident epithelial, stroma and immune 520 cells and the cellular heterogeneity of innervating blood vessels, lymphatics, and peripheral nerves.   In conclusion, a complete whole embryo-fetal cell atlas across human gestation will be challenging 551 but possible in the near future. It will require a global collaborative multi-disciplinary team effort,    The Human Developmental Cell Atlas: how to build it and what will it provide? a. 'How to build an atlas' modules, including an interdisciplinary team, multi-modal technologies, and integration of data across platforms. b. Key features of the Human Development Cell Atlas. Single cell measurements across three dimensional space, alongside a fourth dimension of time, allow for capture of dynamic developmental processes including cell proliferation, migration and regulation. c. Utility and applications of the Human Development Cell Atlas: cellular and molecular biological insights applied to advance regenerative medicine, tissue engineering and therapeutics.

Figure 3
Multi-omics pro ling and data integration a. Organ or anatomical unit pro ling of a prenatal embryo derived from the three germ layers. b. Single cell atlas technologies by relative resolution and genome scale. c. Integration of datasets from different technologies (e.g., spatial transcriptomics, single-cell RNA sequencing, targeted in situ sequencing) to pro le organs or whole embryo.

Supplementary Files
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