Multipotent RAG1+ progenitors emerge directly from haemogenic endothelium in human pluripotent stem cell-derived haematopoietic organoids

Motazedian, Ali; Bruveris, Freya; Kumar, Sanjeev; Schiesser, Jacqueline V.; Chen, Tyrone; Ng, Elizabeth; Chidgey, Ann Patricia; Wells, Christine A.; Elefanty, Andrew G.; Stanley, Edouard G.

doi:10.1038/s41556-019-0445-8

Cited by 43 publications

(41 citation statements)

References 58 publications

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“…Reports comparing mobilization regiments revealed a direct effect of G-CSF on mPB-CD34 + cells, with the proportion of HSCs capable of reconstituting NSG mice being decreased 37 . The diminished capacity of iPSC-CD34 + cells to support the differentiation of T-lineage cells could be attributed to the inability of existing protocols to fully recapitulate the spatial and temporal events occurring during embryonic hematopoiesis 38 – 41 . Strategies to bias lineage choice or generate true HSCs would increase the efficiency of T cell generation 42 – 44 , adapting these approaches to the DL4-μbead platform is of interest and could increase the efficiency of T-lineage generation from PSC.…”

Section: Discussionmentioning

confidence: 99%

DL4-μbeads induce T cell lineage differentiation from stem cells in a stromal cell-free system

et al. 2021

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T cells are pivotal effectors of the immune system and can be harnessed as therapeutics for regenerative medicine and cancer immunotherapy. An unmet challenge in the field is the development of a clinically relevant system that is readily scalable to generate large numbers of T-lineage cells from hematopoietic stem/progenitor cells (HSPCs). Here, we report a stromal cell-free, microbead-based approach that supports the efficient in vitro development of both human progenitor T (proT) cells and T-lineage cells from CD34+cells sourced from cord blood, GCSF-mobilized peripheral blood, and pluripotent stem cells (PSCs). DL4-μbeads, along with lymphopoietic cytokines, induce an ordered sequence of differentiation from CD34+ cells to CD34+CD7+CD5+ proT cells to CD3+αβ T cells. Single-cell RNA sequencing of human PSC-derived proT cells reveals a transcriptional profile similar to the earliest thymocytes found in the embryonic and fetal thymus. Furthermore, the adoptive transfer of CD34+CD7+ proT cells into immunodeficient mice demonstrates efficient thymic engraftment and functional maturation of peripheral T cells. DL4-μbeads provide a simple and robust platform to both study human T cell development and facilitate the development of engineered T cell therapies from renewable sources.

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Section: Discussionmentioning

confidence: 99%

DL4-μbeads induce T cell lineage differentiation from stem cells in a stromal cell-free system

et al. 2021

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“…One such study used scRNAseq of mouse embryonic stem cells undergoing differentiation to better characterize hemangiogenic progenitors as derived from FLK1-positive mesodermal progenitors, and identified SRC kinase as a key regulator of this transition (41). Furthermore, a recent study using scRNA-seq to analyze human pluripotent stem cell-derived hematopoietic organoids suggests that RAG1 could be a novel marker of hemogenic progenitors derived from primordial endothelial cells, as RAG1 is co-expressed with known endothelial/hematopoietic progenitor markers (CD34, VE-Cadherin and CD90) at the single cell level (42).…”

Section: Primordial Endothelial Cell Characterizationmentioning

confidence: 99%

Single Cell Analysis in Vascular Biology

Chavkin

Hirschi

2020

Front. Cardiovasc. Med.

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The ability to quantify DNA, RNA, and protein variations at the single cell level has revolutionized our understanding of cellular heterogeneity within tissues. Via such analyses, individual cells within populations previously thought to be homogeneous can now be delineated into specific subpopulations expressing unique sets of genes, enabling specialized functions. In vascular biology, studies using single cell RNA sequencing have revealed extensive heterogeneity among endothelial and mural cells even within the same vessel, key intermediate cell types that arise during blood and lymphatic vessel development, and cell-type specific responses to disease. Thus, emerging new single cell analysis techniques are enabling vascular biologists to elucidate mechanisms of vascular development, homeostasis, and disease that were previously not possible. In this review, we will provide an overview of single cell analysis methods and highlight recent advances in vascular biology made possible through single cell RNA sequencing.

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“…We recently described a protocol to promote cardiovascular development in gastruloids, which required the addition of VEGF, bFGF, and ascorbic acid to standard gastruloid growth culture conditions (Rossi et al, 2020). As VEGF and bFGF are necessary for hematopoiesis in vitro and in vivo (Daniel et al, 2016; Irion et al, 2010; Kennedy et al, 2007; Mikkola et al, 2003; Motazedian et al, 2020; Pardanaud and Dieterlen-Lièvre, 1999; Sugimura et al, 2017), we hypothesized that these culture conditions could also support the development of the blood lineage. Thus, we mined the scRNA-seq dataset of gastruloids grown in the presence of VEGF and bFGF (Rossi et al, 2020), with a focus on clusters that were on the trajectory from epiblast to endothelial progenitors ( Figure 1A ).…”

Section: Resultsmentioning

confidence: 99%

Gastruloids asin vitromodels of embryonic blood development with spatial and temporal resolution

Rossi

Giger

Huebscher

et al. 2021

Preprint

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Gastruloids are three-dimensional embryonic organoids that reproduce key features of early mammalian development in vitro with unique scalability, accessibility, and spatiotemporal similarity to real embryos. Recently, we adapted gastruloid culture conditions to promote cardiovascular development. In this work, we extended these conditions to capture features of embryonic blood development through a combination of immunophenotyping, detailed transcriptomics analysis, and identification of blood stem/progenitor cell potency. We uncovered the emergence of blood progenitor and erythroid-like cell populations in late gastruloids and showed the multipotent clonogenic capacity of these cells, both in vitro and after transplantation into irradiated mice. We also identified the spatial localization near a vessel-like plexus in the anterior of gastruloids with similarities to the emergence of blood stem cells in the embryo. These results highlight the potential and applicability of gastruloids to the in vitro study of complex processes in embryonic blood development with spatiotemporal fidelity.

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Multipotent RAG1+ progenitors emerge directly from haemogenic endothelium in human pluripotent stem cell-derived haematopoietic organoids

Cited by 43 publications

References 58 publications

DL4-μbeads induce T cell lineage differentiation from stem cells in a stromal cell-free system

DL4-μbeads induce T cell lineage differentiation from stem cells in a stromal cell-free system

Single Cell Analysis in Vascular Biology

Gastruloids asin vitromodels of embryonic blood development with spatial and temporal resolution

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