Mouse studies have been instrumental in forming our current understanding of early cell-lineage decisions; however, similar insights into the early human development are severely limited. Here, we ...present a comprehensive transcriptional map of human embryo development, including the sequenced transcriptomes of 1,529 individual cells from 88 human preimplantation embryos. These data show that cells undergo an intermediate state of co-expression of lineage-specific genes, followed by a concurrent establishment of the trophectoderm, epiblast, and primitive endoderm lineages, which coincide with blastocyst formation. Female cells of all three lineages achieve dosage compensation of X chromosome RNA levels prior to implantation. However, in contrast to the mouse, XIST is transcribed from both alleles throughout the progression of this expression dampening, and X chromosome genes maintain biallelic expression while dosage compensation proceeds. We envision broad utility of this transcriptional atlas in future studies on human development as well as in stem cell research.
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•Transcriptomes of 1,529 individual cells from 88 human preimplantation embryos•Lineage segregation of trophectoderm, primitive endoderm, and pluripotent epiblast•X chromosome dosage compensation in the human blastocyst
A comprehensive transcriptional map of human preimplantation development reveals a concurrent establishment of trophectoderm, epiblast, and primitive endoderm lineages and unique features of X chromosome dosage compensation in human.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Developmental biologists have become increasingly aware that the wealth of knowledge generated through genetic studies of pre-implantation mouse development might not easily be translated to the ...human embryo. Comparative studies have been fueled by recent technological advances in single-cell analysis, allowing in-depth analysis of the human embryo. This field could shortly gain more momentum as novel genome editing technologies might, for the first time, also allow functional genetic studies in the human embryo. In this Spotlight article, we summarize the CRISPR-Cas9 genome editing system and discuss its potential applications and limitations in human pre-implantation embryos, and the ethical considerations thereof.
Human pluripotent stem cells (PSCs) exist in naive and primed states and provide important models to investigate the earliest stages of human development. Naive cells can be obtained through ...primed-to-naive resetting, but there are no reliable methods to prospectively isolate unmodified naive cells during this process. Here we report comprehensive profiling of cell surface proteins by flow cytometry in naive and primed human PSCs. Several naive-specific, but not primed-specific, proteins were also expressed by pluripotent cells in the human preimplantation embryo. The upregulation of naive-specific cell surface proteins during primed-to-naive resetting enabled the isolation and characterization of live naive cells and intermediate cell populations. This analysis revealed distinct transcriptional and X chromosome inactivation changes associated with the early and late stages of naive cell formation. Thus, identification of state-specific proteins provides a robust set of molecular markers to define the human PSC state and allows new insights into the molecular events leading to naive cell resetting.
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•Flow cytometry profiles cell surface proteins in naive and primed human PSCs•The human PSC state can be defined using robust state-specific protein markers•Identified cell surface proteins track the dynamics of naive-primed PSC conversions•Analyses of early-stage naive cells reveal transcription events during conversion
Collier et al. use profiling to identify cell surface proteins that are specific for naive versus primed human pluripotent cells and then use them to isolate and characterize live naive cells arising during primed-to-naive resetting.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Although endonuclease-mediated genome editing techniques offer significant improvement over traditional methods, they are still ineffective for introduction of large DNA sequences. Recently in Nature ...Biotechnology, Gu et al. (2018) developed a CRISPR-Cas strategy termed 2C-HR-CRISPR that generates fluorescent reporter tagging of genes with up to 95% knockin efficiency in mouse embryos.
Although endonuclease-mediated genome editing techniques offer significant improvement over traditional methods, they are still ineffective for introduction of large DNA sequences. Recently in Nature Biotechnology, Gu et al. (2018) developed a CRISPR-Cas strategy termed 2C-HR-CRISPR that generates fluorescent reporter tagging of genes with up to 95% knockin efficiency in mouse embryos.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In vitro differentiation of human pluripotent stem cells into functional retinal pigment epithelial (RPE) cells provides a potentially unlimited source for cell based reparative therapy of ...age-related macular degeneration. Although the inherent pigmentation of the RPE cells have been useful to grossly evaluate differentiation efficiency and allowed manual isolation of pigmented structures, accurate quantification and automated isolation has been challenging. To address this issue, here we perform a comprehensive antibody screening and identify cell surface markers for RPE cells. We show that these markers can be used to isolate RPE cells during in vitro differentiation and to track, quantify and improve differentiation efficiency. Finally, these surface markers aided to develop a robust, direct and scalable monolayer differentiation protocol on human recombinant laminin-111 and -521 without the need for manual isolation.
Functional genetics of early human development Ortega, Nicolás M; Winblad, Nerges; Plaza Reyes, Alvaro ...
Current opinion in genetics & development,
10/2018, Volume:
52
Journal Article
Peer reviewed
Open access
Understanding the genetic underpinning of early human development is of great interest not only for basic developmental and stem cell biology but also for regenerative medicine, infertility ...treatments, and better understanding the causes of congenital disease. Our current knowledge has mainly been generated with the use of laboratory animals, especially the mouse. While human and mouse early development present morphological resemblance, we know that the timing of the events as well as the cellular and genetic mechanisms that control fundamental processes are distinct between the species. The rapid technological development of single-cell sequencing and genome editing together with novel stem cell models of the early human embryo has made it feasible and relevant to perform functional genetic studies directly in human cells and embryos. In this review we will discuss these latest advances where combined transcriptional analysis and genome engineering has begun to shed new insights into the key processes of zygotic genome activation, lineage specification, X-chromosome inactivation and postimplantation development including primordial germ cell specification in the human embryo.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Human embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells could replace lost tissue in geographic atrophy (GA) but efficacy has yet to be demonstrated in a large-eyed model. ...Also, production of hESC-RPE has not yet been achieved in a xeno-free and defined manner, which is critical for clinical compliance and reduced immunogenicity. Here we describe an effective differentiation methodology using human laminin-521 matrix with xeno-free and defined medium. Differentiated cells exhibited characteristics of native RPE including morphology, pigmentation, marker expression, monolayer integrity, and polarization together with phagocytic activity. Furthermore, we established a large-eyed GA model that allowed in vivo imaging of hESC-RPE and host retina. Cells transplanted in suspension showed long-term integration and formed polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity. We have developed a xeno-free and defined hESC-RPE differentiation method and present evidence of functional integration of clinically compliant hESC-RPE in a large-eyed disease model.
•Xeno-free and defined differentiation of hES-RPE cells using recombinant laminin-521•Functional monolayer integration of hES-RPE cells in a novel large-eyed disease model•Rescue of photoreceptors from induced degeneration by transplanted hES-RPE cells
Lanner and colleagues present an effective xeno-free and defined hES-RPE cell differentiation methodology using a recombinant human laminin-521 matrix. They further present evidence of long-term functional integration and photoreceptor rescue by transplanted hES-RPE cells in a large-eyed disease model.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Leucine twenty homeobox (LEUTX) is a paired (PRD)-like homeobox gene that is expressed almost exclusively in human embryos during preimplantation development. We previously identified a novel ...transcription start site for the predicted human LEUTX gene based on the transcriptional analysis of human preimplantation embryos. The novel variant encodes a protein with a complete homeodomain. Here, we provide a detailed description of the molecular cloning of the complete homeodomain-containing LEUTX Using a human embryonic stem cell overexpression model we show that the complete homeodomain isoform is functional and sufficient to activate the transcription of a large proportion of the genes that are upregulated in human embryo genome activation (EGA), whereas the previously predicted partial homeodomain isoform is largely inactive. Another PRD-like transcription factor, DPRX, is then upregulated as a powerful repressor of transcription. We propose a two-stage model of human EGA in which LEUTX acts as a transcriptional activator at the 4-cell stage, and DPRX as a balancing repressor at the 8-cell stage. We conclude that LEUTX is a candidate regulator of human EGA.
As pluripotent stem cell (PSC)‐based reparative cell therapies are reaching the bedside, there is a growing need for the standardization of studies concerning safety of the derived products. Clinical ...trials using these promising strategies are in development, and treatment for age‐related macular degeneration is one of the first that has reached patients. We have previously established a xeno‐free and defined differentiation protocol to generate functional human embryonic stem cells (hESCs)‐derived retinal pigment epithelial (RPE) cells. In this study, we perform preclinical safety studies including karyotype and whole‐genome sequencing (WGS) to assess genome stability, single‐cell RNA sequencing to ensure cell purity, and biodistribution and tumorigenicity analysis to rule out potential migratory or tumorigenic properties of these cells. WGS analysis illustrates that existing germline variants load is higher than the introduced variants acquired through in vitro culture or differentiation, and enforces the importance to examine the genome integrity at a deeper level than just karyotype. Altogether, we provide a strategy for preclinical evaluation of PSC‐based therapies and the data support safety of the hESC‐RPE cells generated through our in vitro differentiation methodology.
Retinal pigment epithelial cells derived from human embryonic stem cells are a promising source for replacement therapy in age‐related macular degeneration. However, preclinical studies concerning safety of the derived products are indispensable. Here, we evaluate safety invitro and invivo, reassuring stability, purity, and lack of tumorigenicity or migratory potential of the derived product for safe clinical translation.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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