Gi-Coupled GPCR Signaling Controls the Formation and Organization of Human Pluripotent Colonies

BACKGROUND:Reprogramming adult human somatic cells to create human induced pluripotent stem (hiPS) cell colonies involves a dramatic morphological and organizational transition. These colonies are morphologically indistinguishable from those of pluripotent human embryonic stem (hES) cells. G protein-coupled receptors (GPCRs) are required in diverse developmental processes, but their role in pluripotent colony morphology and organization is unknown. We tested the hypothesis that G(i)-coupled GPCR signaling contributes to the characteristic morphology and organization of human pluripotent colonies. METHODOLOGY/PRINCIPAL FINDINGS:Specific and irreversible inhibition of G(i)-coupled GPCR signaling by pertussis toxin markedly altered pluripotent colony morphology. Wild-type hES and hiPS cells formed monolayer colonies, but colonies treated with pertussis toxin retracted inward, adopting a dense, multi-layered conformation. The treated colonies were unable to reform after a scratch wound insult, whereas control colonies healed completely within 48 h. In contrast, activation of an alternative GPCR pathway, G(s)-coupled signaling, with cholera toxin did not affect colony morphology or the healing response. Pertussis toxin did not alter the proliferation, apoptosis or pluripotency of pluripotent stem cells. CONCLUSIONS/SIGNIFICANCE:Experiments with pertussis toxin suggest that G(i) signaling plays a critical role in the morphology and organization of pluripotent colonies. These results may be explained by a G(i)-mediated density-sensing mechanism that propels the cells radially outward. GPCRs are a promising target for modulating the formation and organization of hiPS and hES cell colonies and may be important for understanding somatic cell reprogramming and for engineering pluripotent stem cells for therapeutic applications

Induced 2C Expression and Implantation-Competent Blastocyst-like Cysts from Primed Pluripotent Stem Cells

Soon after fertilization, the few totipotent cells of mammalian embryos diverge to form a structure called the blastocyst (BC). Although numerous cell types, including germ cells and extended-pluripotency stem cells, have been developed from pluripotent stem cells (PSCs) in vitro, generating functional BCs only from PSCs remains elusive. Here, we describe induced self-organizing 3D BC-like cysts (iBLCs) generated from mouse PSC culture. Resembling natural BCs, iBLCs have a blastocoel-like cavity and were formed with outer cells expressing trophectoderm lineage markers and with inner cells expressing pluripotency markers. iBLCs transplanted to pseudopregnant mice uteruses implanted, induced decidualization, and exhibited growth and development before resorption, demonstrating that iBLCs are implantation competent. iBLC precursor intermediates required the transcription factor Prdm14 and concomitantly activated the totipotency-related cleavage-stage MERVL reporter and 2C genes. Thus, our system may contribute to the understanding of molecular mechanisms underpinning totipotency, embryogenesis, and implantation

G(i)-coupled GPCR signaling controls the formation and organization of human pluripotent colonies.

BackgroundReprogramming adult human somatic cells to create human induced pluripotent stem (hiPS) cell colonies involves a dramatic morphological and organizational transition. These colonies are morphologically indistinguishable from those of pluripotent human embryonic stem (hES) cells. G protein-coupled receptors (GPCRs) are required in diverse developmental processes, but their role in pluripotent colony morphology and organization is unknown. We tested the hypothesis that G(i)-coupled GPCR signaling contributes to the characteristic morphology and organization of human pluripotent colonies.Methodology/principal findingsSpecific and irreversible inhibition of G(i)-coupled GPCR signaling by pertussis toxin markedly altered pluripotent colony morphology. Wild-type hES and hiPS cells formed monolayer colonies, but colonies treated with pertussis toxin retracted inward, adopting a dense, multi-layered conformation. The treated colonies were unable to reform after a scratch wound insult, whereas control colonies healed completely within 48 h. In contrast, activation of an alternative GPCR pathway, G(s)-coupled signaling, with cholera toxin did not affect colony morphology or the healing response. Pertussis toxin did not alter the proliferation, apoptosis or pluripotency of pluripotent stem cells.Conclusions/significanceExperiments with pertussis toxin suggest that G(i) signaling plays a critical role in the morphology and organization of pluripotent colonies. These results may be explained by a G(i)-mediated density-sensing mechanism that propels the cells radially outward. GPCRs are a promising target for modulating the formation and organization of hiPS and hES cell colonies and may be important for understanding somatic cell reprogramming and for engineering pluripotent stem cells for therapeutic applications

Non-thermal atmospheric-pressure plasma potentiates mesodermal differentiation of human induced pluripotent stem cells

Non-thermal atmospheric-pressure plasma has been used for biological applications, including sterilization and stimulation of cell growth and differentiation. Here, we demonstrate that plasma exposure influences the differentiation pattern of human induced pluripotent stem cells (hiPSCs). We treated hiPSCs with dielectric barrier-discharge air plasma and found an exposure dose that does not kill hiPSCs. Immunohistochemical staining for E-CADHERIN showed that the exposure affected cell-cell attachment and doubled the average size of the hiPSCs. Analysis of mRNAs in embryoid bodies (EBs) from plasma-treated hiPSCs revealed repression of ectoderm genes, including WNT1, and increased expression of mesoderm genes. Importantly, hiPSCs deficient in DNA repair only displayed minimal damage after plasma exposure. Collectively, our results suggest that plasma treatment can be another tool for directing the fate of pluripotent stem cells without disrupting their genomic integrity

Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors

Successful reprogramming of differentiated human somatic cells into a pluripotent state would allow creation of patient- and disease-specific stem cells. We previously reported generation of induced pluripotent stem (iPS) cells, capable of germline transmission, from mouse somatic cells by transduction of four defined transcription factors. Here, we demonstrate the generation of iPS cells from adult human dermal fibroblasts with the same four factors: Oct3/4, Sox2, Klf4, and c-Myc. Human iPS cells were similar to human embryonic stem (ES) cells in morphology, proliferation, surface antigens, gene expression, epigenetic status of pluripotent cell-specific genes, and telomerase activity. Furthermore, these cells could differentiate into cell types of the three germ layers in vitro and in teratomas. These findings demonstrate that iPS cells can be generated from adult human fibroblasts

Autotaxin-mediated lipid signaling intersects with LIF and BMP signaling to promote the naive pluripotency transcription factor program

Developmental signaling molecules are used for cell fate determination, and understanding how their combinatorial effects produce the variety of cell types in multicellular organisms is a key problem in biology. Here, we demonstrate that the combination of leukemia inhibitory factor (LIF), bone morphogenetic protein 4 (BMP4), lysophosphatidic acid (LPA), and ascorbic acid (AA) efficiently converts mouse primed pluripotent stem cells (PSCs) into naive PSCs. Signaling by the lipid LPA through its receptor LPAR1 and downstream effector Rho-associated protein kinase (ROCK) cooperated with LIF signaling to promote this conversion. BMP4, which also stimulates conversion to naive pluripotency, bypassed the need for exogenous LPA by increasing the activity of the extracellular LPA-producing enzyme autotaxin (ATX). We found that LIF and LPA-LPAR1 signaling affect the abundance of signal transducer and activator of transcription 3 (STAT3), which induces a previously unappreciated Kruppel-like factor (KLF)2-KLF4-PR domain 14 (PRDM14) transcription factor circuit key to establish naive pluripotency. AA also affects this transcription factor circuit by controlling PRDM14 expression. Thus, our study reveals that ATX-mediated autocrine lipid signaling promotes naive pluripotency by intersecting with LIF and BMP4 signaling

Autotaxin-mediated lipid signaling intersects with LIF and BMP signaling to promote the naive pluripotency transcription factor program

Developmental signaling molecules are used for cell fate determination, and understanding how their combinatorial effects produce the variety of cell types in multicellular organisms is a key problem in biology. Here, we demonstrate that the combination of leukemia inhibitory factor (LIF), bone morphogenetic protein 4 (BMP4), lysophosphatidic acid (LPA), and ascorbic acid (AA) efficiently converts mouse primed pluripotent stem cells (PSCs) into naive PSCs. Signaling by the lipid LPA through its receptor LPAR1 and downstream effector Rho-associated protein kinase (ROCK) cooperated with LIF signaling to promote this conversion. BMP4, which also stimulates conversion to naive pluripotency, bypassed the need for exogenous LPA by increasing the activity of the extracellular LPA-producing enzyme autotaxin (ATX). We found that LIF and LPA-LPAR1 signaling affect the abundance of signal transducer and activator of transcription 3 (STAT3), which induces a previously unappreciated Kruppel-like factor (KLF)2-KLF4-PR domain 14 (PRDM14) transcription factor circuit key to establish naive pluripotency. AA also affects this transcription factor circuit by controlling PRDM14 expression. Thus, our study reveals that ATX-mediated autocrine lipid signaling promotes naive pluripotency by intersecting with LIF and BMP4 signaling

ACVR1R206H extends inflammatory responses in human induced pluripotent stem cell-derived macrophages

Macrophages play crucial roles in many human disease processes. However, obtaining large numbers of primary cells for study is often difficult. We describe 2D and 3D methods for directing human induced pluripotent stem cells (hiPSCs) into macrophages (iMACs). iMACs generated in 2D culture showed functional similarities to human primary monocyte-derived M2-like macrophages, and could be successfully polarized into a M1-like phenotype. Both M1- and M2-like iMACs showed phagocytic activity and reactivity to endogenous or exogenous stimuli. In contrast, iMACs generated by a 3D culture system showed mixed M1- and M2-like functional characteristics. 2D-iMACs from patients with fibrodysplasia ossificans progressiva (FOP), an inherited disease with progressive heterotopic ossification driven by inflammation, showed prolonged inflammatory cytokine production and higher Activin A production after M1-like polarization, resulting in dampened responses to additional LPS stimulation. These results demonstrate a simple and robust way of creating hiPSC-derived M1- and M2-like macrophage lineages, while identifying macrophages as a source of Activin A that may drive heterotopic ossification in FOP