Connexin 43 Gene Ablation Does Not Alter Human Pluripotent Stem Cell Germ Lineage Specification

During embryonic germ layer development, cells communicate with each other and their environment to ensure proper lineage specification and tissue development. Connexin (Cx) proteins facilitate direct cell–cell communication through gap junction channels. While previous reports suggest that gap junctional intercellular communication may contribute to germ layer formation, there have been limited comprehensive expression analyses or genetic ablation studies on Cxs during human pluripotent stem cell (PSC) germ lineage specification. We screened the mRNA profile and protein expression patterns of select human Cx isoforms in undifferentiated human induced pluripotent stem cells (iPSCs), and after directed differentiation into the three embryonic germ lineages: ectoderm, definitive endoderm, and mesoderm. Transcript analyses by qPCR revealed upregulation of Cx45 and Cx62 in iPSC-derived ectoderm; Cx45 in mesoderm; and Cx30.3, Cx31, Cx32, Cx36, Cx37, and Cx40 in endoderm relative to control human iPSCs. Generated Cx43 (GJA1) CRISPR-Cas9 knockout iPSCs successfully differentiated into cells of all three germ layers, suggesting that Cx43 is dispensable during directed iPSC lineage specification. Furthermore, qPCR screening of select Cx transcripts in our GJA1-/- iPSCs showed no significant Cx upregulation in response to the loss of Cx43 protein. Future studies will reveal possible compensation by additional Cxs, suggesting targets for future CRISPR-Cas9 ablation studies in human iPSC lineage specification.

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Spatio-temporal re-organization of replication foci accompanies replication domain consolidation during human pluripotent stem cell lineage specification

Cell Cycle ◽

10.1080/15384101.2016.1203492 ◽

2016 ◽

Vol 15 (18) ◽

pp. 2464-2475 ◽

Cited By ~ 20

Author(s):

Korey A. Wilson ◽

Andrew G. Elefanty ◽

Edouard G. Stanley ◽

David M. Gilbert

Keyword(s):

Stem Cell ◽

Pluripotent Stem Cell ◽

Cell Lineage ◽

Human Pluripotent Stem Cell ◽

Lineage Specification ◽

Stem Cell Lineage ◽

Replication Domain ◽

Replication Foci ◽

Spatio Temporal

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Gene regulatory network (GRN) embedded agents connect cellular decision making to human pluripotent stem cell derived germ layer-like pattern formation

10.1101/2020.10.06.327650 ◽

2020 ◽

Author(s):

Himanshu Kaul ◽

Nicolas Werschler ◽

Mukul Tewary ◽

Andrew Hagner ◽

Joel Ostblom ◽

...

Keyword(s):

Stem Cell ◽

Gene Regulatory Network ◽

Regulatory Network ◽

Regulatory Networks ◽

Pluripotent Stem Cell ◽

Network Activity ◽

Germ Layer ◽

Human Pluripotent Stem Cell ◽

Gene Regulatory

ABSTRACTThe emergence of germ layers in embryos during gastrulation is a key developmental milestone. How morphogenetic signals engage the regulatory networks responsible for early embryonic tissue patterning is incompletely understood. To understand this, we developed a gene regulatory network (GRN) model of human pluripotent stem cell (hPSC) lineage commitment and embedded it into ‘cellular’ agents that respond to a dynamic signalling microenvironment. We found that cellular pattern order, composition, and dynamics were predictably manipulable based on the GRN wiring. We showed that feedback between OCT4, and BMP and WNT pathways created a dynamic OCT4 front that mediates the spatiotemporal evolution of developmental patterns. Translocation of this radial front can be predictively disrupted in vitro to control germ-layer pattern composition. This work links the emergence of multicellular patterns to regulatory network activity in individual hPSCs. We anticipate our approach will help to understand how GRN structure regulates organogenesis in different contexts.

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Pluripotent Stem Cell-derived Cerebral Organoids Reveal Human Oligodendrogenesis with Dorsal and Ventral Origins

10.1101/460907 ◽

2018 ◽

Cited By ~ 1

Author(s):

Hyosung Kim ◽

Ranjie Xu ◽

Padmashri Ragunathan ◽

Anna Dunaevsky ◽

Ying Liu ◽

...

Keyword(s):

Stem Cell ◽

Human Brain ◽

Pluripotent Stem Cell ◽

Expression Patterns ◽

Neuronal Maturation ◽

Human Pluripotent Stem Cell ◽

Temporal Expression ◽

Functional Differences ◽

Rodent Brain

SUMMARYThe process of oligodendrogenesis has been relatively well delineated in the rodent brain. However, it remains unknown whether analogous developmental processes are manifested in the human brain. Here, we report oligodendrogenesis in forebrain organoids, generated by using OLIG2-GFP knockin human pluripotent stem cell (hPSC) reporter lines. OLIG2/GFP exhibits distinct temporal expression patterns in ventral forebrain organoids (VFOs) vs. dorsal forebrain organoids (DFOs). Interestingly, oligodendrogenesis can be induced in both VFOs and DFOs after neuronal maturation. Assembling VFOs and DFOs to generate fused forebrain organoids (FFOs) promotes oligodendroglia maturation. Furthermore, dorsally-derived oligodendroglial cells outcompete ventrally-derived oligodendroglia and become dominant in FFOs after long-term culture. Thus, our organoid models reveal human oligodendrogenesis with ventral and dorsal origins. These models will serve to study the phenotypic and functional differences between human ventrally- and dorsally-derived oligodendroglia and to reveal mechanisms of diseases associated with cortical myelin defects.

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