Project description:Stem cell-derived tissues have wide potential for modelling developmental and pathological processes as well as cell-based therapy. However, it has proven difficult to generate several key cell types in vitro, including skeletal muscle. In vertebrates, skeletal muscles derive during embryogenesis from the presomitic mesoderm (PSM). Using PSM development as a guide, we establish conditions for the differentiation of monolayer cultures of human pluripotent stem (hPSC) cells into PSM-like cells without the introduction of transgenes or cell sorting. We differentiated human PSCs in serum-free medium supplemented with Chir99021 only (C medium) or with also the Bmp inhibitor LDN193189 (CL medium). In vivo, the PSM cells are first expressing MSGN1 (posterior PSM marker) and then mature to express Pax3 (anterior PSM marker). After 4-5 days of differentiation of hPSCs, MSGN1-positive cells were FACS-sorted and their transcriptome analyzed.
Project description:Stem cell-derived tissues have wide potential for modelling developmental and pathological processes as well as cell-based therapy. However, it has proven difficult to generate several key cell types in vitro, including skeletal muscle. In vertebrates, skeletal muscles derive during embryogenesis from the presomitic mesoderm (PSM). Using PSM development as a guide, we establish conditions for the differentiation of monolayer cultures of mouse embryonic stem (ES) cells into PSM-like cells without the introduction of transgenes or cell sorting. We differentiated mouse ESCs in serum-free medium supplemented with Rspo3 ( or as an alternative with Chir 9902) and the Bmp inhibitor LDN193189. In vivo, the PSM cells are first expressing Msgn1 (posterior PSM marker) and then mature to express Pax3 (anterior PSM marker). After 4 days of differentiation of mESCs, Msgn1-positive cells were FACS-sorted and their transcriptome analyzed. After 6 days of differentiation, Pax3-positive cells were sorted and their transcriptome analyzed. Mouse ESCs differentiated for 0, 4 and 6 days in serum-free medium containing a Wnt activator, a BMP inhibitor and DMSO, to study paraxial mesoderm in vitro
Project description:During the development of the vertebrate embryo, segmented structures called somites are periodically formed from the presomitic mesoderm (PSM), and give rise to the vertebral column. While somite formation has been studied in several animal models, it is less clear how well this process is conserved in humans. Recent progress has made it possible to study aspects of human paraxial mesoderm development such as the human segmentation clock in vitro using human pluripotent stem cells (hPSCs), however, somite formation has not been observed in these monolayer cultures. Here, we describe the generation of human paraxial mesoderm (PM) organoids from hPSCs (termed Somitoids), which recapitulate the molecular, morphological and functional features of paraxial mesoderm development, including formation of somite-like structures in vitro. Using a quantitative image-based screen, we identify critical parameters such as initial cell number and signaling modulations that reproducibly yielded somite formation in our organoid system. In addition, using single-cell RNA sequencing and 3D imaging, we show that PM organoids both transcriptionally and morphologically resemble their in vivo counterparts and can be differentiated into somite derivatives. Our organoid system is reproducible and scalable, allowing for the systematic and quantitative analysis of human spinal cord development and disease in vitro.
Project description:The trunk axial skeleton develops from paraxial mesoderm cells. Our recent study demonstrated that conditional knockout of the stem cell factor Sall4 in mice by TCre caused tail truncation and a disorganized axial skeleton posterior to the lumbar level. Based on this phenotype, we hypothesized that, in addition to the previously reported role of Sall4 in neuromesodermal progenitors, Sall4 is involved in the development of the paraxial mesoderm tissue. ATAC-seq in TCre; Sall4 mutant posterior trunk mesoderm shows that Sall4 knockout reduces chromatin accessibility. We found that Sall4- dependent open chromatin status drives activation and repression of WNT signaling activators and repressors, respectively, to promote WNT signaling. Moreover, footprinting analysis of ATAC-seq data suggests that Sall4-dependent chromatin accessibility facilitates CTCF binding, which contributes to the repression of neural genes within the mesoderm. This study unveils multiple mechanisms by which Sall4 regulates paraxial mesoderm development by directing activation of mesodermal genes and repression of neural genes.
Project description:Laser capture microdissection (LCM) was used to isolate cells from the principal critical micro-regions, whose development, differentiation and signaling interactions are responsible for the construction of the mammalian face. At E8.5, as migrating neural crest cells begin to exit the neural fold/epidermal ectoderm boundary, we examined the facial mesenchyme, composed of neural crest and paraxial mesoderm cells, as well as cells from adjacent neuroepithelium We performed single cell studies to better define the gene expression states of the early E8.5 pioneer neural crest cells and paraxial mesoderm, and present microarray data detailing expression patterns within these embryonic cell populations. Mouse emrbyos were harvested at developmental stage E8.5 and single cells were captured from the neuroepithilium, neural crest, and paraxial mesoderm. RNA was extracted, labelled, and quantified using the Mouse ST-l microarray.
Project description:Brown adipocytes (BAs) are a potential therapeutic cell source for the treatment of metabolic disease such as type 2 diabetes. In this report, human pluripotent stem cells (hPSCs) are subject to directed differentiation to brown dipocytes through a paraxial mesoderm intermediate at high-efficiency. RNA-Seq and ATAC-seq was performed to characterized hPSCs derived paraxial mesoderm and brown adipocytes generated in this study.
Project description:Stem cell-derived tissues have wide potential for modelling developmental and pathological processes as well as cell-based therapy. However, it has proven difficult to generate several key cell types in vitro, including skeletal muscle. In vertebrates, skeletal muscles derive during embryogenesis from the presomitic mesoderm (PSM). Using PSM development as a guide, we establish conditions for the differentiation of monolayer cultures of mouse embryonic stem (ES) cells into PSM-like cells without the introduction of transgenes or cell sorting. We differentiated mouse ESCs in serum-free medium supplemented with Rspo3 ( or as an alternative with Chir 9902) and the Bmp inhibitor LDN193189. In vivo, the PSM cells are first expressing Msgn1 (posterior PSM marker) and then mature to express Pax3 (anterior PSM marker). After 4 days of differentiation of mESCs, Msgn1-positive cells were FACS-sorted and their transcriptome analyzed. After 6 days of differentiation, Pax3-positive cells were sorted and their transcriptome analyzed.
Project description:Cardiac development requires precise gene expression programs at each developmental stage guided by multiple signaling pathways and transcription factors (TFs). MESP1 is transiently expressed in mesoderm, and is essential for subsequent cardiac development, while the precise mechanism regulating its own transcription and mesoderm cell fate is not fully understood. Therefore, we developed a high content screen assay to identify regulators of MESP1 expression in mesodermal cells differentiated from human pluripotent stem cells (hPSCs). The screen identified CYT387, a JAK1/JAK2 kinase inhibitor, as a potent molecule that can significantly increase MESP1 expression. CYT387 was also found to enhance cardiomyocyte differentiation from hPSCs in vitro. Mechanistical studies found that JAK inhibition promotes MESP1 expression by reducing cytoplasmic calcium concentration and subsequently activating canonical WNT signaling. Our study identified a role of JAK signaling in early mesoderm cells, and sheds light on the connection between the JAK-STAT pathway and transcriptional regulation of MESP1, which expands our understanding of mesoderm and cardiac development.