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Vogt, Nina. "Making limb-like structures from mouse PSCs". Nature Methods 16, nr 10 (27.09.2019): 957. http://dx.doi.org/10.1038/s41592-019-0603-8.
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Tan, Zenglai, Aleksandra Rak-Raszewska, Ilya Skovorodkin i Seppo J. Vainio. "Mouse Embryonic Stem Cell-Derived Ureteric Bud Progenitors Induce Nephrogenesis". Cells 9, nr 2 (31.01.2020): 329. http://dx.doi.org/10.3390/cells9020329.
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Generation of kidney organoids from pluripotent stem cells (PSCs) is regarded as a potentially powerful way to study kidney development, disease, and regeneration. Direct differentiation of PSCs towards renal lineages is well studied; however, most of the studies relate to generation of nephron progenitor population from PSCs. Until now, differentiation of PSCs into ureteric bud (UB) progenitor cells has had limited success. Here, we describe a simple, efficient, and reproducible protocol to direct differentiation of mouse embryonic stem cells (mESCs) into UB progenitor cells. The mESC-derived UB cells were able to induce nephrogenesis when co-cultured with primary metanephric mesenchyme (pMM). In generated kidney organoids, the embryonic pMM developed nephron structures, and the mESC-derived UB cells formed numerous collecting ducts connected with the nephron tubules. Altogether, our study established an uncomplicated and reproducible platform to generate ureteric bud progenitors from mouse embryonic stem cells.
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Wang, Xuepeng, i Qiang Wu. "The Divergent Pluripotent States in Mouse and Human Cells". Genes 13, nr 8 (16.08.2022): 1459. http://dx.doi.org/10.3390/genes13081459.
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Pluripotent stem cells (PSCs), which can self-renew and give rise to all cell types in all three germ layers, have great potential in regenerative medicine. Recent studies have shown that PSCs can have three distinct but interrelated pluripotent states: naive, formative, and primed. The PSCs of each state are derived from different stages of the early developing embryo and can be maintained in culture by different molecular mechanisms. In this review, we summarize the current understanding on features of the three pluripotent states and review the underlying molecular mechanisms of maintaining their identities. Lastly, we discuss the interrelation and transition among these pluripotency states. We believe that comprehending the divergence of pluripotent states is essential to fully harness the great potential of stem cells in regenerative medicine.
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FRECH, MORITZ J., i KURT H. BACKUS. "Characterization of inhibitory postsynaptic currents in rod bipolar cells of the mouse retina". Visual Neuroscience 21, nr 4 (lipiec 2004): 645–52. http://dx.doi.org/10.1017/s0952523804214134.
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The synaptic terminals of mammalian rod bipolar cells are the targets of multiple presynaptic inhibitory inputs arriving from glycinergic and GABAergic amacrine cells. To investigate the contribution of these different inhibitory receptor types, we have applied the patch-clamp technique in acutely isolated slices of the adult mouse retina. By using the whole-cell configuration, we measured and analyzed the spontaneous postsynaptic currents (PSCs) in rod bipolar cells. The spontaneous synaptic activity of rod bipolar cells was very low. However, when amacrine cells were depolarized by AMPA or kainate, the PSC frequency in rod bipolar cells increased significantly. These PSCs comprised several types that could be distinguished by pharmacological and kinetic criteria. Strychnine-sensitive, glycinergic PSCs were characterized by a mean peak amplitude of −43.5 pA and a weighted decay time constant (τw) of 10.9 ms. PSCs that persisted in the presence of strychnine, but were completely inhibited by bicuculline, were mediated by GABAARs. They had a mean peak amplitude of −20.0 pA and a significantly faster τwof 5.8 ms. Few PSCs remained in the presence of strychnine and bicuculline, suggesting that they were mediated by GABACRs. These PSCs were characterized by much smaller amplitudes (−6.2 pA) and a significantly slower decay kinetics (τw= 51.0 ms). We conclude that rod bipolar cells express at least three types of functionally different inhibitory receptors, namely GABAARs, GABACRs, and GlyRs that may ultimately regulate the Ca2+influx into rod bipolar cell terminals, thereby modulating their glutamate release.
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Menzorov, Aleksei G. "Pluripotent Stem Cells of Order Carnivora: Technical Perspective". International Journal of Molecular Sciences 24, nr 4 (15.02.2023): 3905. http://dx.doi.org/10.3390/ijms24043905.
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Human and mouse induced pluripotent stem cells (PSCs) are widely used for studying early embryonic development and for modeling of human diseases. Derivation and studying of PSCs from model organisms beyond commonly used mice and rats may provide new insights into the modeling and treating human diseases. The order Carnivora representatives possess unique features and are already used for modeling human-related traits. This review focuses on the technical aspects of derivation of the Carnivora species PSCs as well as their characterization. Current data on dog, feline, ferret, and American mink PSCs are summarized.
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Barazangi, Nobl, i Lorna W. Role. "Nicotine-Induced Enhancement of Glutamatergic and GABAergic Synaptic Transmission in the Mouse Amygdala". Journal of Neurophysiology 86, nr 1 (1.07.2001): 463–74. http://dx.doi.org/10.1152/jn.2001.86.1.463.
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Presynaptic nicotinic acetylcholine receptors (nAChRs) are thought to mediate some of the cognitive and behavioral effects of nicotine. The olfactory projection to the amygdala, and intra-amygdaloid projections, are limbic relays involved in behavioral reinforcement, a property influenced by nicotine. Co-cultures consisting of murine olfactory bulb (OB) explants and dispersed amygdala neurons were developed to reconstruct this pathway in vitro. Whole cell patch-clamp recordings were obtained from amygdala neurons contacted by OB explant neurites, and spontaneous and evoked synaptic currents were characterized. The majority of the 108 innervated amygdala neurons exhibited glutamatergic spontaneous postsynaptic currents (PSCs), 20% exhibited GABAergic spontaneous PSCs, and 17% exhibited both. Direct extracellular stimulation of OB explants elicited glutamatergic synaptic currents in amygdala neurons. Antibodies to nAChR subunits co-localized with an antibody to synapsin I, a presynaptic marker, along OB explant processes, consistent with the targeting of nAChR protein to presynaptic sites of the mitral cell projections. Hence, we examined the role of presynaptic nAChRs in modulating synaptic transmission in the OB–amygdala co-cultures. Focal application of 500 nM to 1 μM nicotine for 5–60 s markedly increased the frequency of spontaneous PSCs, without a change in the amplitude, in 39% of neurons that exhibited glutamatergic spontaneous PSCs (average peak fold increase = 125.2 ± 33.3). Nicotine also enhanced evoked glutamatergic currents elicited by direct stimulation of OB explant fibers. Nicotine increased the frequency of spontaneous PSCs, without a change in the amplitude, in 35% of neurons that exhibited GABAergic spontaneous PSCs (average peak fold increase = 63.9 ± 34.3). Thus activation of presynaptic nAChRs can modulate glutamatergic as well as GABAergic synaptic transmission in the amygdala. These results suggest that behaviors mediated by olfactory projections may be modulated by presynaptic nAChRs in the amygdala, where integration of olfactory and pheromonal input is thought to occur.
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Li, Jiahui, Xiaolin Wu, Lars Schiffmann, Thomas MacVicar, Chenghui Zhou, Zhefang Wang, Dai Li i in. "IL-17B/RB Activation in Pancreatic Stellate Cells Promotes Pancreatic Cancer Metabolism and Growth". Cancers 13, nr 21 (24.10.2021): 5338. http://dx.doi.org/10.3390/cancers13215338.
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In pancreatic ductal adenocarcinoma (PDAC), the tumor stroma constitutes most of the cell mass and contributes to therapy resistance and progression. Here we show a hitherto unknown metabolic cooperation between pancreatic stellate cells (PSCs) and tumor cells through Interleukin 17B/Interleukin 17B receptor (IL-17B/IL-17RB) signaling. Tumor-derived IL-17B carrying extracellular vesicles (EVs) activated stromal PSCs and induced the expression of IL-17RB. PSCs increased oxidative phosphorylation while reducing mitochondrial turnover. PSCs activated tumor cells in a feedback loop. Tumor cells subsequently increased oxidative phosphorylation and decreased glycolysis partially via IL-6. In vivo, IL-17RB overexpression in PSCs accelerated tumor growth in a co-injection xenograft mouse model. Our results demonstrate a tumor-to-stroma feedback loop increasing tumor metabolism to accelerate tumor growth under optimal nutritional conditions.
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Ng, Benjamin, Sivakumar Viswanathan, Anissa A. Widjaja, Wei-Wen Lim, Shamini G. Shekeran, Joyce Wei Ting Goh, Jessie Tan i in. "IL11 Activates Pancreatic Stellate Cells and Causes Pancreatic Inflammation, Fibrosis and Atrophy in a Mouse Model of Pancreatitis". International Journal of Molecular Sciences 23, nr 7 (24.03.2022): 3549. http://dx.doi.org/10.3390/ijms23073549.
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Interleukin-11 (IL11) is important for fibrosis and inflammation, but its role in the pancreas is unclear. In pancreatitis, fibrosis, inflammation and organ dysfunction are associated with pancreatic stellate cell (PSC)-to-myofibroblast transformation. Here, we show that IL11 stimulation of PSCs, which specifically express IL11RA in the pancreas, results in transient STAT3 phosphorylation, sustained ERK activation and PSC activation. In contrast, IL6 stimulation of PSCs caused sustained STAT3 phosphorylation but did not result in ERK activation or PSC transformation. Pancreatitis factors, including TGFβ, CTGF and PDGF, induced IL11 secretion from PSCs and a neutralising IL11RA antibody prevented PSC activation by these stimuli. This revealed an important ERK-dependent role for autocrine IL11 activity in PSCs. In mice, IL11 was increased in the pancreas after pancreatic duct ligation, and in humans, IL11 and IL11RA levels were elevated in chronic pancreatitis. Following pancreatic duct ligation, administration of anti-IL11RA to mice reduced pathologic (ERK, STAT, NF-κB) signalling, pancreatic atrophy, fibrosis and pro-inflammatory cytokine (TNFα, IL6 and IL1β) levels. This is the first description of IL11-mediated activation of PSCs, and the data suggest IL11 as a stromal therapeutic target in pancreatitis.
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Zhou, Zhen, Xiaodong Sun, Rao Yan, Jinfeng An, Xinjian Zhou, Mingyi Li, Xinsheng Gu, Xincai Hao i Ming Sang. "Resveratrol inhibits high glucose-induced activation and cytokine production of isolated primary pancreatic stellate cells". Journal of Applied Virology 8, nr 3 (8.12.2019): 35–47. http://dx.doi.org/10.21092/jav.v8i3.112.
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Objective: Activation of pancreatic stellate cells (PSCs) is detrimental to pancreas function by promoting pancreatic fibrosis. Resveratrol is a natural and pharmacologically active compound. This study is to investigate the effect of resveratrol on the bilogical behavior of PSCs under high glucose condition.Methods: Isolated primary mouse PSCs were cultured in low glucose ( 5.5 mmol/L glucose, LG group ) medium, high glucose ( 25 mmol/L glucose, HG group ) medium and treated with resveratrol ( 25 μmol/L or 50 μmol/L). Cell proliferation was examined using MTT assay. The expression of α-SMA and collagen I were determined using Western blotting. Alpha-SMA expression was also determined using immunocytochemistry staining. IL-1, IL-6, and TNF-α mRNA levels and secretion levels in media of PSCs were determined using qRT-PCR and ELISA respectively.Results: Cell Proliferation, α-SMA and collagen I expression levels, IL-1, IL-6, and TNF-α mRNA levels and secretion levels of PSCs were increased after high glucose treatment, compared with low glucose treatment. They were significantly decreased in PSCs treated with both high glucose and resveratrol, compared with high glucose treatment.Conclusion: Resveratrol inhibited high glucose induced PSCs proliferation, activation，cytokine expression and secretion in PSCs. Therefore, resveratrol can be potentially used in therapy of diseases such as type 2 diabetes mellitus (T2DM), pancreatitis and pancreatic cancer where PSCs is activated by high glucose.
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Jolly, Gurbani, Tetyana Duka, Narayan Shivapurkar, Wenqiang Chen, Sunil Bansal, Amrita Cheema i Jill P. Smith. "Cholecystokinin Receptor Antagonist Induces Pancreatic Stellate Cell Plasticity Rendering the Tumor Microenvironment Less Oncogenic". Cancers 15, nr 10 (18.05.2023): 2811. http://dx.doi.org/10.3390/cancers15102811.
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CCK receptors are expressed on pancreatic cancer epithelial cells, and blockade with receptor antagonists decreases tumor growth. Activated pancreatic stellate cells or myofibroblasts have also been described to express CCK receptors, but the contribution of this novel pathway in fibrosis of the pancreatic cancer microenvironment has not been studied. We examined the effects of the nonselective CCK receptor antagonist proglumide on the activation, proliferation, collagen deposition, differential expression of genes, and migration in both murine and human PSCs. CCK receptor expression was examined using western blot analysis. Collagen production using activated PSCs was analyzed by mass spectroscopy and western blot. Migration of activated PSCs was prevented in vitro by proglumide and the CCK-B receptor antagonist, L365,260, but not by the CCK-A receptor antagonist L365,718. Proglumide effectively decreased the expression of extracellular matrix-associated genes and collagen-associated proteins in both mouse and human PSCs. Components of fibrosis, including hydroxyproline and proline levels, were significantly reduced in PSC treated with proglumide compared to controls. CCK peptide stimulated mouse and human PSC proliferation, and this effect was blocked by proglumide. These investigations demonstrate that targeting the CCK-B receptor signaling pathway with proglumide may alter the plasticity of PSC, rendering them more quiescent and leading to a decrease in fibrosis in the pancreatic cancer microenvironment.
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Uchida, Chiaki, Hiroki Mizukami, Yutaro Hara, Takeshi Saito, Satoko Umetsu, Akiko Igawa, Sho Osonoi i in. "Diabetes in Humans Activates Pancreatic Stellate Cells via RAGE in Pancreatic Ductal Adenocarcinoma". International Journal of Molecular Sciences 22, nr 21 (28.10.2021): 11716. http://dx.doi.org/10.3390/ijms222111716.
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Pancreatic stellate cells (PSCs) mainly consist of cancer-associating fibroblasts in pancreatic ductal adenocarcinoma (PDAC). The receptor for advanced glycation end products (RAGE) is implicated in the pathophysiology of diabetic complications. Here, we studied the implication of RAGE in PSC activation in PDAC. The activation of cultured mouse PSCs was evaluated by qPCR. The induction of epithelial mesenchymal transition (EMT) in PDAC cell lines was assessed under stimulation with culture supernatant from activated PSCs. A total of 155 surgically resected PDAC subjects (83 nondiabetic, 18 with ≦3-years and 54 with >3-years history of diabetes) were clinicopathologically evaluated. A high-fat diet increased the expression of activated markers in cultured PSCs, which was abrogated by RAGE deletion. Culture supernatant from activated PSCs facilitated EMT of PDAC cells with elevation of TGF−β and IL−6, but not from RAGE−deleted PSCs. Diabetic subjects complicated with metabolic syndrome, divided by cluster analysis, showed higher PSC activation and RAGE expression. In such groups, PDAC cells exhibited an EMT nature. The complication of metabolic syndrome with diabetes significantly worsened disease−free survival of PDAC subjects. Thus, RAGE in PSCs can be viewed as a new promoter and a future therapeutic target of PDAC in diabetic subjects with metabolic syndrome.
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Pappas, Matthew P., Ning Xie, Jacqueline S. Penaloza i Sunny S. K. Chan. "Defining the Skeletal Myogenic Lineage in Human Pluripotent Stem Cell-Derived Teratomas". Cells 11, nr 9 (9.05.2022): 1589. http://dx.doi.org/10.3390/cells11091589.
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Skeletal muscle stem cells are essential to muscle homeostasis and regeneration after injury, and have emerged as a promising cell source for treating skeletal disorders. An attractive approach to obtain these cells utilizes differentiation of pluripotent stem cells (PSCs). We recently reported that teratomas derived from mouse PSCs are a rich source of skeletal muscle stem cells. Here, we showed that teratoma formation is also capable of producing skeletal myogenic progenitors from human PSCs. Using single-cell transcriptomics, we discovered several distinct skeletal myogenic subpopulations that represent progressive developmental stages of the skeletal myogenic lineage and recapitulate human embryonic skeletal myogenesis. We further discovered that ERBB3 and CD82 are effective surface markers for prospective isolation of the skeletal myogenic lineage in human PSC-derived teratomas. Therefore, teratoma formation provides an accessible model for obtaining human skeletal myogenic progenitors from PSCs.
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Panova, A. V., E. D. Nekrasov, M. A. Lagarkova, S. L. Kiselev i A. N. Bogomazova. "Late Replication of the Inactive X Chromosome Is Independent of the Compactness of Chromosome Territory in Human Pluripotent Stem Cells". Acta Naturae 5, nr 2 (15.06.2013): 54–61. http://dx.doi.org/10.32607/20758251-2013-5-2-54-61.
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Dosage compensation of the X chromosomes in mammals is performed via the formation of facultative heterochromatin on extra X chromosomes in female somatic cells. Facultative heterochromatin of the inactivated X (Xi), as well as constitutive heterochromatin, replicates late during the S-phase. It is generally accepted that Xi is always more compact in the interphase nucleus. The dense chromosomal folding has been proposed to define the late replication of Xi. In contrast to mouse pluripotent stem cells (PSCs), the status of X chromosome inactivation in human PSCs may vary significantly. Fluorescence in situ hybridization with a whole X-chromosome-specific DNA probe revealed that late-replicating Xi may occupy either compact or dispersed territory in human PSCs. Thus, the late replication of the Xi does not depend on the compactness of chromosome territory in human PSCs. However, the Xi reactivation and the synchronization in the replication timing of X chromosomes upon reprogramming are necessarily accompanied by the expansion of X chromosome territory.
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Devika, A. S., Anna Montebaur, S. Saravanan, Raghu Bhushan, Frederic Koch i Smita Sudheer. "Human ES Cell Culture Conditions Fail to Preserve the Mouse Epiblast State". Stem Cells International 2021 (10.03.2021): 1–12. http://dx.doi.org/10.1155/2021/8818356.
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Mouse embryonic stem cells (mESCs) and mouse epiblast stem cells (mEpiSCs) are the pluripotent stem cells (PSCs), derived from the inner cell mass (ICM) of preimplantation embryos at embryonic day 3.5 (E3.5) and postimplantation embryos at E5.5-E7.5, respectively. Depending on their environment, PSCs can exist in the so-called naïve (ESCs) or primed (EpiSCs) states. Exposure to EpiSC or human ESC (hESC) culture condition can convert mESCs towards an EpiSC-like state. Here, we show that the undifferentiated epiblast state is however not stabilized in a sustained manner when exposing mESCs to hESC or EpiSC culture condition. Rather, prolonged exposure to EpiSC condition promotes a transition to a primitive streak- (PS-) like state via an unbiased epiblast-like intermediate. We show that the Brachyury-positive PS-like state is likely promoted by endogenous WNT signaling, highlighting a possible species difference between mouse epiblast-like stem cells and human Embryonic Stem Cells.
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Astle, John, Yangfei Xiang, Anthony Rongvaux, Carla Weibel, Henchey Elizabeth, Stephanie Halene, In-Hyun Park i Richard Flavell. "Developing a Model of Human Pluripotent to Hematopoietic Stem Cell Development in Mistrg Mice". Blood 126, nr 23 (3.12.2015): 4755. http://dx.doi.org/10.1182/blood.v126.23.4755.4755.
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Abstract De novo generation of HSCs has been described as a "holy grail" of stem cell biology, however the factors required for converting human pluripotent stem cells (PSCs) to true hematopoietic stem cells (HSCs) capable of robust long-term engraftment have yet to be fully characterized. Two groups have shown that injection of PSCs into immunodeficient mice leads to teratomas containing niches producing hematopoietic progenitors capable of long-term engraftment. Once these hematopoietic progenitors and their microenvironments are better characterized, this system could be used as a model to help direct in vitro differentiation of PSCs to HSCs. Toward this end, we have injected human PSCs into immunodeficient mice expressing human rather than mouse M-CSF, IL-3, GM-CSF, and thrombopoietin, as well as both human and mouse versions of the "don't eat me signal" Sirpa (collectively termed MISTRG mice). These cytokines are known to support different aspects of hematopoiesis, and thrombopoietin in particular has been shown to support HSC maintenance, suggesting these mice may provide a better environment for human PSC-derived HSCs than the more traditional mice used for human HSC engraftment. The majority of teratomas developed so far in MISTRG contain human hematopoietic cells, and the CD34+ population isolated from over half of the teratomas contained hematopoietic colony forming cells by colony forming assay. These findings further corroborate this approach as a viable method for studying human PSC to HSC differentiation. Disclosures No relevant conflicts of interest to declare.
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Tani, Shoichiro, Ung-il Chung, Shinsuke Ohba i Hironori Hojo. "Understanding paraxial mesoderm development and sclerotome specification for skeletal repair". Experimental & Molecular Medicine 52, nr 8 (sierpień 2020): 1166–77. http://dx.doi.org/10.1038/s12276-020-0482-1.
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Abstract Pluripotent stem cells (PSCs) are attractive regenerative therapy tools for skeletal tissues. However, a deep understanding of skeletal development is required in order to model this development with PSCs, and for the application of PSCs in clinical settings. Skeletal tissues originate from three types of cell populations: the paraxial mesoderm, lateral plate mesoderm, and neural crest. The paraxial mesoderm gives rise to the sclerotome mainly through somitogenesis. In this process, key developmental processes, including initiation of the segmentation clock, formation of the determination front, and the mesenchymal–epithelial transition, are sequentially coordinated. The sclerotome further forms vertebral columns and contributes to various other tissues, such as tendons, vessels (including the dorsal aorta), and even meninges. To understand the molecular mechanisms underlying these developmental processes, extensive studies have been conducted. These studies have demonstrated that a gradient of activities involving multiple signaling pathways specify the embryonic axis and induce cell-type-specific master transcription factors in a spatiotemporal manner. Moreover, applying the knowledge of mesoderm development, researchers have attempted to recapitulate the in vivo development processes in in vitro settings, using mouse and human PSCs. In this review, we summarize the state-of-the-art understanding of mesoderm development and in vitro modeling of mesoderm development using PSCs. We also discuss future perspectives on the use of PSCs to generate skeletal tissues for basic research and clinical applications.
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Świerczek-Lasek, Barbara, Damian Dudka, Damian Bauer, Tomasz Czajkowski, Katarzyna Ilach, Władysława Streminska, Agata Kominek, Katarzyna Piwocka, Maria A. Ciemerych i Karolina Archacka. "Comparison of Differentiation Pattern and WNT/SHH Signaling in Pluripotent Stem Cells Cultured under Different Conditions". Cells 10, nr 10 (14.10.2021): 2743. http://dx.doi.org/10.3390/cells10102743.
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Pluripotent stem cells (PSCs) are characterized by the ability to self-renew as well as undergo multidirectional differentiation. Culture conditions have a pivotal influence on differentiation pattern. In the current study, we compared the fate of mouse PSCs using two culture media: (1) chemically defined, free of animal reagents, and (2) standard one relying on the serum supplementation. Moreover, we assessed the influence of selected regulators (WNTs, SHH) on PSC differentiation. We showed that the differentiation pattern of PSCs cultured in both systems differed significantly: cells cultured in chemically defined medium preferentially underwent ectodermal conversion while their endo- and mesodermal differentiation was limited, contrary to cells cultured in serum-supplemented medium. More efficient ectodermal differentiation of PSCs cultured in chemically defined medium correlated with higher activity of SHH pathway while endodermal and mesodermal conversion of cells cultured in serum-supplemented medium with higher activity of WNT/JNK pathway. However, inhibition of either canonical or noncanonical WNT pathway resulted in the limitation of endo- and mesodermal conversion of PSCs. In addition, blocking WNT secretion led to the inhibition of PSC mesodermal differentiation, confirming the pivotal role of WNT signaling in this process. In contrast, SHH turned out to be an inducer of PSC ectodermal, not mesodermal differentiation.
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Świerczek-Lasek, Barbara, Damian Dudka, Damian Bauer, Tomasz Czajkowski, Katarzyna Ilach, Władysława Streminska, Agata Kominek, Katarzyna Piwocka, Maria A. Ciemerych i Karolina Archacka. "Comparison of Differentiation Pattern and WNT/SHH Signaling in Pluripotent Stem Cells Cultured under Different Conditions". Cells 10, nr 10 (14.10.2021): 2743. http://dx.doi.org/10.3390/cells10102743.
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Pluripotent stem cells (PSCs) are characterized by the ability to self-renew as well as undergo multidirectional differentiation. Culture conditions have a pivotal influence on differentiation pattern. In the current study, we compared the fate of mouse PSCs using two culture media: (1) chemically defined, free of animal reagents, and (2) standard one relying on the serum supplementation. Moreover, we assessed the influence of selected regulators (WNTs, SHH) on PSC differentiation. We showed that the differentiation pattern of PSCs cultured in both systems differed significantly: cells cultured in chemically defined medium preferentially underwent ectodermal conversion while their endo- and mesodermal differentiation was limited, contrary to cells cultured in serum-supplemented medium. More efficient ectodermal differentiation of PSCs cultured in chemically defined medium correlated with higher activity of SHH pathway while endodermal and mesodermal conversion of cells cultured in serum-supplemented medium with higher activity of WNT/JNK pathway. However, inhibition of either canonical or noncanonical WNT pathway resulted in the limitation of endo- and mesodermal conversion of PSCs. In addition, blocking WNT secretion led to the inhibition of PSC mesodermal differentiation, confirming the pivotal role of WNT signaling in this process. In contrast, SHH turned out to be an inducer of PSC ectodermal, not mesodermal differentiation.
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Takahashi, Yuuwa, i Shogo Miyata. "Continuous ES/Feeder Cell-Sorting Device Using Dielectrophoresis and Controlled Fluid Flow". Micromachines 11, nr 8 (29.07.2020): 734. http://dx.doi.org/10.3390/mi11080734.
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Pluripotent stem cells (PSCs) are considered as being an important cell source for regenerative medicine. The culture of PSCs usually requires a feeder cell layer or cell adhesive matrix coating such as Matrigel, laminin, and gelatin. Although a feeder-free culture using a matrix coating has been popular, the on-feeder culture is still an effective method for the fundamental study of regenerative medicine and stem cell biology. To culture PSCs on feeder cell layers, the elimination of feeder cells is required for biological or gene analysis and for cell passage. Therefore, a simple and cost-effective cell sorting technology is required. There are several commercialized cell-sorting methods, such as FACS or MACS. However, these methods require cell labeling by fluorescent dye or magnetic antibodies with complicated processes. To resolve these problems, we focused on dielectrophoresis (DEP) phenomena for cell separation because these do not require any fluorescent or magnetic dyes or antibodies. DEP imposes an electric force on living cells under a non-uniform AC electric field. The direction and magnitude of the DEP force depend on the electric property and size of the cell. Therefore, DEP is considered as a promising approach for sorting PSCs from feeder cells. In this study, we developed a simple continuous cell-sorting device using the DEP force and fluid-induced shear force. As a result, mouse embryonic stem cells (mESCs) were purified from a mixed-cell suspension containing mESCs and mouse embryonic fibroblasts (MEFs) using our DEP cell-sorting device.
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Wang, Shao-Hua, Chao Zhang i Yangming Wang. "microRNA regulation of pluripotent state transition". Essays in Biochemistry 64, nr 6 (grudzień 2020): 947–54. http://dx.doi.org/10.1042/ebc20200028.
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Abstract microRNAs (miRNAs) play essential roles in mouse embryonic stem cells (ESCs) and early embryo development. The exact mechanism by which miRNAs regulate cell fate transition during embryo development is still not clear. Recent studies have identified and captured various pluripotent stem cells (PSCs) that share similar characteristics with cells from different stages of pre- and post-implantation embryos. These PSCs provide valuable models to understand miRNA functions in early mammalian development. In this short review, we will summarize recent work towards understanding the function and mechanism of miRNAs in regulating the transition or conversion between different pluripotent states. In addition, we will highlight unresolved questions and key future directions related to miRNAs in pluripotent state transition. Studies in these areas will further our understanding of miRNA functions in early embryo development, and may lead to practical means to control human PSCs for clinical applications in regenerative medicine.
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Che, Mingtian, Soo-Mi Kweon, Jia-Ling Teo, Yate-Ching Yuan, Laleh G. Melstrom, Richard T. Waldron, Aurelia Lugea, Raul A. Urrutia, Stephen J. Pandol i Keane K. Y. Lai. "Targeting the CBP/β-Catenin Interaction to Suppress Activation of Cancer-Promoting Pancreatic Stellate Cells". Cancers 12, nr 6 (5.06.2020): 1476. http://dx.doi.org/10.3390/cancers12061476.
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Background: Although cyclic AMP-response element binding protein-binding protein (CBP)/β-catenin signaling is known to promote proliferation and fibrosis in various organ systems, its role in the activation of pancreatic stellate cells (PSCs), the key effector cells of desmoplasia in pancreatic cancer and fibrosis in chronic pancreatitis, is largely unknown. Methods: To investigate the role of the CBP/β-catenin signaling pathway in the activation of PSCs, we have treated mouse and human PSCs with the small molecule specific CBP/β-catenin antagonist ICG-001 and examined the effects of treatment on parameters of activation. Results: We report for the first time that CBP/β-catenin antagonism suppresses activation of PSCs as evidenced by their decreased proliferation, down-regulation of “activation” markers, e.g., α-smooth muscle actin (α-SMA/Acta2), collagen type I alpha 1 (Col1a1), Prolyl 4-hydroxylase, and Survivin, up-regulation of peroxisome proliferator activated receptor gamma (Ppar-γ) which is associated with quiescence, and reduced migration; additionally, CBP/β-catenin antagonism also suppresses PSC-induced migration of cancer cells. Conclusion: CBP/β-catenin antagonism represents a novel therapeutic strategy for suppressing PSC activation and may be effective at countering PSC promotion of pancreatic cancer.
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Xiao, Ying, Tao Qin, Liankang Sun, Weikun Qian, Jie Li, Wanxing Duan, Jianjun Lei i in. "Resveratrol Ameliorates the Malignant Progression of Pancreatic Cancer by Inhibiting Hypoxia-induced Pancreatic Stellate Cell Activation". Cell Transplantation 29 (1.01.2020): 096368972092998. http://dx.doi.org/10.1177/0963689720929987.
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Pancreatic cancer is characterized by a hypoxic tumor microenvironment, which is primarily caused by massive fibrosis with pancreatic stellate cells (PSCs) as a main component. Our previous studies have shown that resveratrol can significantly inhibit pancreatic cancer. However, whether resveratrol can inhibit hypoxia-induced cancer development remains unclear. The objective of this study was to explore whether PSCs and hypoxia synergistically mediate aggressiveness in pancreatic cancer and detect the potential pleiotropic protective effects of resveratrol on hypoxia-induced pancreatic cancer progression. Human PSCs were treated with vehicle or resveratrol under normoxic or hypoxic conditions (3% O2), and PSC activation was assessed by immunofluorescence staining. SiRNA was used to silence hypoxia-inducible factor 1 (HIF-1) expression. The invasive capacity of Panc-1 and Mia Paca-2 cells cocultured with conditioned medium from PSCs was assessed by Transwell assays. To examine tumor formation kinetics, KPC (LSL-KrasG12D/+, Trp53fl/+, and Pdx1-Cre) mice were sacrificed at different time points. To investigate the antitumor effects of resveratrol in vivo, 8-wk-old KPC mice were divided into two groups and treated daily with or without 50 mg/kg resveratrol. Our data indicate that hypoxia induces PSC activation via HIF-1 and that the interleukin 6, vascular endothelial growth factor A, and stromal cell-derived factor 1 derived from activated PSCs promote both invasion and the epithelial–mesenchymal transition and inhibit apoptosis in pancreatic cancer cells. However, resveratrol inhibits hypoxia-induced PSC activation, blocks the interplay between PSCs and pancreatic cancer cells, and suppresses the malignant progression of pancreatic cancer and stromal desmoplasia in a KPC mouse model. Our data highlight that activated PSCs and intratumoral hypoxia are essential targets for novel strategies to prevent tumor–microenvironment interactions. Furthermore, the polyphenolic compound resveratrol effectively ameliorates the malignant progression of pancreatic ductal adenocarcinoma.
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Madsen, Ralitsa R. "PI3K in stemness regulation: from development to cancer". Biochemical Society Transactions 48, nr 1 (3.02.2020): 301–15. http://dx.doi.org/10.1042/bst20190778.
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The PI3K/AKT pathway is a key target in oncology where most efforts are focussed on phenotypes such as cell proliferation and survival. Comparatively, little attention has been paid to PI3K in stemness regulation, despite the emerging link between acquisition of stem cell-like features and therapeutic failure in cancer. The aim of this review is to summarise current known and unknowns of PI3K-dependent stemness regulation, by integrating knowledge from the fields of developmental, signalling and cancer biology. Particular attention is given to the role of the PI3K pathway in pluripotent stem cells (PSCs) and the emerging parallels to dedifferentiated cancer cells with stem cell-like features. Compelling evidence suggests that PI3K/AKT signalling forms part of a ‘core molecular stemness programme’ in both mouse and human PSCs. In cancer, the oncogenic PIK3CAH1047R variant causes constitutive activation of the PI3K pathway and has recently been linked to increased stemness in a dose-dependent manner, similar to observations in mouse PSCs with heterozygous versus homozygous Pten loss. There is also evidence that the stemness phenotype may become ‘locked’ and thus independent of the original PI3K activation, posing limitations for the success of PI3K monotherapy in cancer. Ongoing therapeutic developments for PI3K-associated cancers may therefore benefit from a better understanding of the pathway's two-layered and highly context-dependent regulation of cell growth versus stemness.
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Öhlund, Daniel, Abram Handly-Santana, Giulia Biffi, Ela Elyada, Ana S. Almeida, Mariano Ponz-Sarvise, Vincenzo Corbo i in. "Distinct populations of inflammatory fibroblasts and myofibroblasts in pancreatic cancer". Journal of Experimental Medicine 214, nr 3 (23.02.2017): 579–96. http://dx.doi.org/10.1084/jem.20162024.
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Pancreatic stellate cells (PSCs) differentiate into cancer-associated fibroblasts (CAFs) that produce desmoplastic stroma, thereby modulating disease progression and therapeutic response in pancreatic ductal adenocarcinoma (PDA). However, it is unknown whether CAFs uniformly carry out these tasks or if subtypes of CAFs with distinct phenotypes in PDA exist. We identified a CAF subpopulation with elevated expression of α-smooth muscle actin (αSMA) located immediately adjacent to neoplastic cells in mouse and human PDA tissue. We recapitulated this finding in co-cultures of murine PSCs and PDA organoids, and demonstrated that organoid-activated CAFs produced desmoplastic stroma. The co-cultures showed cooperative interactions and revealed another distinct subpopulation of CAFs, located more distantly from neoplastic cells, which lacked elevated αSMA expression and instead secreted IL6 and additional inflammatory mediators. These findings were corroborated in mouse and human PDA tissue, providing direct evidence for CAF heterogeneity in PDA tumor biology with implications for disease etiology and therapeutic development.
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Manshaei, Saba, Thea L. Willis, Virinder Reen, Husayn Pallikonda, Jodie Birch, Dominic J. Withers, Jesus Gil, Cynthia L. Andoniadou i Juan Pedro Martinez-Barbera. "RF13 | PMON143 BRF1-Mediated Paracrine Signalling by a Subset of SOX2-Expressing Stem Cells is Required for Normal Development of the Stem Cell Compartment and Terminal Differentiation of Pituitary Committed Progenitors". Journal of the Endocrine Society 6, Supplement_1 (1.11.2022): A580—A581. http://dx.doi.org/10.1210/jendso/bvac150.1203.
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Abstract Introduction Hormone-producing pituitary cell lineages are derived from a population of embryonic precursors expressing SOX2. These cells maintain multipotency into early postnatal life, acting as the resident population of pituitary stem cells (PSCs) and contributing extensively to all the endocrine cell lineages. In addition to this direct contribution to pituitary turnover, paracrine signalling from PSCs has been shown to be important for cell proliferation of neighbouring progenitors (PMC7803373). It is not known if SOX2+ PSCs are involved in the regulation of additional cell attributes during normal physiology and if there is functional heterogeneity among the SOX2+ PSC population. Experimental Methods We have carried out single-cell RNA-Sequencing of SOX2+ PSCs from Sox2Egfp/+ mouse pituitaries at three postnatal stages from P3 to P56 and used computational approaches to analyse their molecular signatures. A novel conditional mouse model expressing a constitutively active mutant form of the RNA binding factor BRF1 (R26stop-mBRF1) has been used to attenuate the expression of several cytokines and chemokines in SOX2+ cells embryonically and postnatally (PMC4589897). Results We show that the SOX2+ PSC population consists of three subgroups (SC1, SC2 and SC3). We reveal that SC1-SC2 express abundant cytokines and secreted factors, suggesting paracrine function. In contrast, SC3 is characterised by robust expression of Lef1, is identified as a committing PSC cluster, and its presence diminishes with age. Key markers of PSC clusters SC1-SC2 include the RNA binding factor BRF1. We show that BRF1 is highly expressed in PSCs and validate its expression by immunohistochemistry in both mouse and human pituitaries. Secondly, we show that the dysregulation of BRF1 in embryonic SOX2+ cells using the Hesx1-Cre driver (PMC3461924) results in pituitary hypoplasia and severe hypopituitarism due to a failure of the PIT1 and SF1 cell-lineage committed progenitors to terminally differentiate into hormone-producing cells. Additionally, there is a significant reduction of the stem cell compartment, manifested by lower numbers of SOX2/SOX9+ stem cells. This phenotype is recapitulated when using a Sox2-CreERT2 driver (PMID24094324). The differentiation failure can be rescued in vitro through co-culture of mutant cells with wild-type stem cells, as well as in vivo, in mutant pituitaries where activation of constitutively active BRF1 is restricted to few SOX2+ PSCs in a mosaic manner. Finally, we identify key ligands underlying this differentiation phenotype and demonstrate a partial restoration of terminal differentiation in the mutant, when cultured in the presence of these ligands. Conclusion We provide evidence indicating the presence of functionally distinct groups of SOX2+ pituitary stem cells and reveal a critical role for a PSC subset in the development of the stem cell compartment and in driving terminal differentiation of committed progenitors. Presentation: Sunday, June 12, 2022 1:00 p.m. - 1:05 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.
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Xie, Ning, Sabrina N. Chu, Cassandra B. Schultz i Sunny S. K. Chan. "Efficient Muscle Regeneration by Human PSC-Derived CD82+ ERBB3+ NGFR+ Skeletal Myogenic Progenitors". Cells 12, nr 3 (18.01.2023): 362. http://dx.doi.org/10.3390/cells12030362.
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Differentiation of pluripotent stem cells (PSCs) is a promising approach to obtaining large quantities of skeletal myogenic progenitors for disease modeling and cell-based therapy. However, generating skeletal myogenic cells with high regenerative potential is still challenging. We recently reported that skeletal myogenic progenitors generated from mouse PSC-derived teratomas possess robust regenerative potency. We have also found that teratomas derived from human PSCs contain a skeletal myogenic population. Here, we showed that these human PSC-derived skeletal myogenic progenitors had exceptional engraftability. A combination of cell surface markers, CD82, ERBB3, and NGFR enabled efficient purification of skeletal myogenic progenitors. These cells expressed PAX7 and were able to differentiate into MHC+ multinucleated myotubes. We further discovered that these cells are expandable in vitro. Upon transplantation, the expanded cells formed new dystrophin+ fibers that reconstituted almost ¾ of the total muscle volume, and repopulated the muscle stem cell pool. Our study, therefore, demonstrates the possibility of producing large quantities of engraftable skeletal myogenic cells from human PSCs.
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Alcalde, Alejandra Diaz, Edoardo Vallariello, Elena Astanina, Emanuele Middonti i Federico Bussolino. "Abstract 2355: Transcription factor EB modulates fibrotic response in pancreatic ductal adenocarcinoma". Cancer Research 83, nr 7_Supplement (4.04.2023): 2355. http://dx.doi.org/10.1158/1538-7445.am2023-2355.
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Abstract Pancreatic ductal adenocarcinoma (PDAC), which comprises 85% of all the pancreatic cancers, is one of the most aggressive and deadly tumor that exists today with less than 8% of survival 5 years after diagnosis. It is predicted to become the second cause of cancer death by 2030. A typical characteristic of this tumor is the presence of prominent stroma component mainly produced by Pancreatic Stellate Cells (PSCs) in response to soluble factors released by cancer cells. PSCs activation and differentiation into CAF (Cancer-associated fibroblasts) is a reversible process, which impacts on both tumor progression and drug resistance. Transcription factor EB (TFEB) is mostly known as a master regulator of autophagy and lysosomal biogenesis. A recent study demonstrated that TFEB inhibits epithelial-to-mesenchymal transition (EMT) and myofibroblast differentiation in epicardial cells by upregulating TGIF1, a TGFβ pathway repressor. Based on this observation, we investigated if TFEB might have an inhibitory role in PSCs activation, taking into consideration the crucial role of TGFβ signaling in this process. The TFEB expression, measured by immunostaining, was significantly downregulated in CAFs in human PDAC samples compared to PSCs in healthy pancreas. In PSCs, co-cultured with PDAC cell lines, CAF markers were upregulated while TFEB expression was downregulated. The overexpression of TFEB in PSCs suppressed CAF markers upregulation induced by co-culture with PDAC cells at both mRNA and protein levels. The bulk RNAseq analysis showed the differential expression of genes required for extracellular matrix production, fibrotic response and inflammation in PSCs co-cultured with PDAC cell lines versus PSCs alone, with and without TFEB overexpression. These in vitro data suggest a role for TFEB in PSCs activation and stroma formation which will be further investigated in PDAC mouse models. Citation Format: Alejandra Diaz Alcalde, Edoardo Vallariello, Elena Astanina, Emanuele Middonti, Federico Bussolino. Transcription factor EB modulates fibrotic response in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2355.
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Haque, Mohammad Shamsul. "Targeting stem cell-derived viral antigen-specific T lymphocytes for HBV immunotherapy". Journal of Immunology 198, nr 1_Supplement (1.05.2017): 78.13. http://dx.doi.org/10.4049/jimmunol.198.supp.78.13.
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Abstract Adoptive cell transfer (ACT) of hepatitis B virus (HBV)-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for chronic HBV infection. Naive or central memory T cell-derived effector CTLs known as “highly reactive” cells are optimal populations for cell-based therapies. However, such ACT is often not feasible due to difficulties in obtaining sufficient numbers of CTLs from patients. Pluripotent stem cells (PSCs) have the capacity to be reprogrammed into functional viral antigen (Ag)-specific CTLs, which have a potential to be used for ACT-based immunotherapy. However, the optimal approach to differentiate viral Ag -specific CTLs from PSCs remains unclear. In this study, we generated HBV-specific CTLs from induced PSCs (iPSCs), i.e., iPSC-CTLs, and utilized a mouse model of chronic HBV infection to test ACT of iPSC-CTLs for HBV immunotherapy. Murine iPSCs that genetically modified with HBV-specific T cell receptor (TCR) were co-cultured with OP9 cell line expressing Notch ligands Delta-like 1 and 4 (OP9-DL1/DL4 cell) for a week and adoptively transferred into recipient C67BL/6 mice for in vivo development. The combination of Notch signaling and incorporation of HBV-specific TCR genes in iPSCs drove the development of HBV-specific iPSC-CTLs. In addition, In a mouse model of chronic HBV infection in which the HBV viral genome is delivered into the mouse liver through hydrodynamic injection, HBV-specific iPSC-CTLs infiltrated into liver tissue, and inhibited HBV replication after ACT. These results confirm that ACT of viral Ag-specific iPSC-CTLs can substantially suppress HBV replication, and indicate that stem cell-derived highly reactive viral Ag-specific CTLs may be used for HBV immunotherapy.
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Xue, Ran, Jianxin Wang, Lixin Yang, Xinjuan Liu, Yan Gao, Yanhua Pang, Yanbin Wang i Jianyu Hao. "Coenzyme Q10 Ameliorates Pancreatic Fibrosis via the ROS-Triggered mTOR Signaling Pathway". Oxidative Medicine and Cellular Longevity 2019 (7.02.2019): 1–10. http://dx.doi.org/10.1155/2019/8039694.
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Aim. Pancreatic stellate cells (PSCs) play a pivotal role in pancreatic fibrosis. Any remedies that inhibit the activation of PSCs can be potential candidates for therapeutic strategies in pancreatic fibrosis-related pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis (CP). Our study is aimed at exploring the protective effect of coenzyme Q10 (CoQ10) against pancreatic fibrosis. Methods. Pancreatic fibrosis was induced by 20% L-arginine (250 mg/100 g) at 1 h intervals twice per week for 8 weeks in C57BL/6 mice. CoQ10 was administered for 4 weeks. Isolated primary PSCs from C57BL/6 mice were treated with 100 μM CoQ10 for 72 h, as well as Rosup and specific inhibitors. The effects of CoQ10 on the activation of PSCs, autophagy, collagen deposition, histological changes, and oxidative stress were analyzed by western blotting, biochemical estimations, immunofluorescence staining, and hematoxylin-eosin, Masson, and Sirius red staining, as well as with a reactive oxygen species (ROS) assay. Results. Pretreatment and posttreatment of CoQ10 decreased autophagy, activation of PSCs, oxidative stress, histological changes, and collagen deposition in the CP mouse model. In primary PSCs, expression levels of p-PI3K, p-AKT, and p-mTOR were upregulated with CoQ10. A rescue experiment using specific inhibitors of the PI3K-AKT-mTOR pathway demonstrated that the PI3K-AKT-mTOR signaling pathway was the underlying mechanism by which CoQ10 ameliorated fibrosis. With the addition of Rosup, expression levels of the autophagy biomarkers LC3 and Atg5 were elevated. Meanwhile, the levels of p-PI3K, p-AKT, and p-mTOR were lower. Conclusions. Our findings demonstrated that CoQ10 alleviates pancreatic fibrosis by the ROS-triggered PI3K/AKT/mTOR signaling pathway. CoQ10 may be a therapeutic candidate for antifibrotic methods.
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Pei, Yangli, Liang Yue, Wei Zhang, Jinzhu Xiang, Zhu Ma i Jianyong Han. "Murine pluripotent stem cells that escape differentiation inside teratomas maintain pluripotency". PeerJ 6 (4.01.2018): e4177. http://dx.doi.org/10.7717/peerj.4177.
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Background Pluripotent stem cells (PSCs) offer immense potential as a source for regenerative therapies. The teratoma assay is widely used in the field of stem cells and regenerative medicine, but the cell composition of teratoma is still elusive. Methods We utilized PSCs expressing enhanced green fluorescent protein (EGFP) under the control of the Pou5f1 promoter to study the persistence of potential pluripotent cells during teratoma formation in vivo. OCT4-MES (mouse embryonic stem cells) were isolated from the blastocysts of 3.5-day OCT4-EGFP mice (transgenic mice express EGFP cDNA under the control of the Pou5f1 promoter) embryos, and TG iPS 1-7 (induced pluripotent stem cells) were generated from mouse embryonic fibroblasts (MEFs) from 13.5-day OCT4-EGFP mice embryos by infecting them with a virus carrying OCT4, SOX2, KLF4 and c-MYC. These pluripotent cells were characterized according to their morphology and expression of pluripotency markers. Their differentiation ability was studied with in vivo teratoma formation assays. Further differences between pluripotent cells were examined by real-time quantitative PCR (qPCR). Results The results showed that several OCT4-expressing PSCs escaped differentiation inside of teratomas, and these escaped cells (MES-FT, GFP-positive cells separated from OCT4-MES-derived teratomas; and iPS-FT, GFP-positive cells obtained from teratomas formed by TG iPS 1-7) retained their pluripotency. Interestingly, a small number of GFP-positive cells in teratomas formed by MES-FT and iPS-FT (MES-ST, GFP-positive cells isolated from MES-FT-derived teratomas; iPS-ST, GFP-positive cells obtained from teratomas formed by iPS-FT) were still pluripotent, as shown by alkaline phosphatase (AP) staining, immunofluorescent staining and PCR. MES-FT, iPS-FT, MES-ST and iPS-ST cells also expressed several markers associated with germ cell formation, such as Dazl, Stella and Stra8. Conclusions In summary, a small number of PSCs escaped differentiation inside of teratomas, and these cells maintained pluripotency and partially developed towards germ cells. Both escaped PSCs and germ cells present a risk of tumor formation. Therefore, medical workers must be careful in preventing tumor formation when stem cells are used to treat specific diseases.
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Easley, Charles A., Calvin R. Simerly i Gerald Schatten. "Gamete derivation from embryonic stem cells, induced pluripotent stem cells or somatic cell nuclear transfer-derived embryonic stem cells: state of the art". Reproduction, Fertility and Development 27, nr 1 (2015): 89. http://dx.doi.org/10.1071/rd14317.
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Generating gametes from pluripotent stem cells (PSCs) has many scientific justifications and several biomedical rationales. Here, we consider several strategies for deriving gametes from PSCs from mice and primates (human and non-human) and their anticipated strengths, challenges and limitations. Although the ‘Weismann barrier’, which separates the mortal somatic cell lineages from the potentially immortal germline, has long existed, breakthroughs first in mice and now in humans are artificially creating germ cells from somatic cells. Spermatozoa with full reproductive viability establishing multiple generations of seemingly normal offspring have been reported in mice and, in humans, haploid spermatids with correct parent-of-origin imprints have been obtained. Similar progress with making oocytes has been published using mouse PSCs differentiated in vitro into primordial germ cells, which are then cultured after xenografting reconstructed artificial ovaries. Progress in making human oocytes artificially is proving challenging. The usefulness of these artificial gametes, from assessing environmental exposure toxicity to optimising medical treatments to prevent negative off-target effects on fertility, may prove invaluable, as may basic discoveries on the fundamental mechanisms of gametogenesis.
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Pang, Tony C. Y., Zhihong Xu, Alpha Raj Mekapogu, Srinivasa Pothula, Therese Becker, Susan Corley, Marc R. Wilkins i in. "HGF/c-Met Inhibition as Adjuvant Therapy Improves Outcomes in an Orthotopic Mouse Model of Pancreatic Cancer". Cancers 13, nr 11 (2.06.2021): 2763. http://dx.doi.org/10.3390/cancers13112763.
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Background: Inhibition of hepatocyte growth factor (HGF)/c-MET pathway, a major mediator of pancreatic stellate cell (PSC)−PC cell interactions, retards local and distant cancer progression. This study examines the use of this treatment in preventing PC progression after resection. We further investigate the postulated existence of circulating PSCs (cPSCs) as a mediator of metastatic PC. Methods: Two orthotopic PC mouse models, produced by implantation of a mixture of luciferase-tagged human pancreatic cancer cells (AsPC-1), and human PSCs were used. Model 1 mice underwent distal pancreatectomy 3-weeks post-implantation (n = 62). One-week post-resection, mice were randomised to four treatments of 8 weeks: (i) IgG, (ii) gemcitabine (G), (iii) HGF/c-MET inhibition (HiCi) and (iv) HiCi + G. Tumour burden was assessed longitudinally by bioluminescence. Circulating tumour cells and cPSCs were enriched by filtration. Tumours of Model 2 mice progressed for 8 weeks prior to the collection of primary tumour, metastases and blood for single-cell RNA-sequencing (scRNA-seq). Results: HiCi treatments: (1) reduced both the risk and rate of disease progression after resection; (2) demonstrated an anti-angiogenic effect on immunohistochemistry; (3) reduced cPSC counts. cPSCs were identified using immunocytochemistry (α-smooth muscle actin+, pan-cytokeratin−, CD45−), and by specific PSC markers. scRNA-seq confirmed the existence of cPSCs and identified potential genes associated with development into cPSCs. Conclusions: This study is the first to demonstrate the efficacy of adjuvant HGF/c-Met inhibition for PC and provides the first confirmation of the existence of circulating PSCs.
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Oikawa, Mami, Hisato Kobayashi, Makoto Sanbo, Naoaki Mizuno, Kenyu Iwatsuki, Tomoya Takashima, Keiko Yamauchi i in. "Functional primordial germ cell–like cells from pluripotent stem cells in rats". Science 376, nr 6589 (8.04.2022): 176–79. http://dx.doi.org/10.1126/science.abl4412.
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The in vitro generation of germ cells from pluripotent stem cells (PSCs) can have a substantial effect on future reproductive medicine and animal breeding. A decade ago, in vitro gametogenesis was established in the mouse. However, induction of primordial germ cell–like cells (PGCLCs) to produce gametes has not been achieved in any other species. Here, we demonstrate the induction of functional PGCLCs from rat PSCs. We show that epiblast-like cells in floating aggregates form rat PGCLCs. The gonadal somatic cells support maturation and epigenetic reprogramming of the PGCLCs. When rat PGCLCs are transplanted into the seminiferous tubules of germline-less rats, functional spermatids—that is, those capable of siring viable offspring—are generated. Insights from our rat model will elucidate conserved and divergent mechanisms essential for the broad applicability of in vitro gametogenesis.
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Ivanyuk, Dina, Galina Budash, Yunjie Zheng, John Antony Gaspar, Umesh Chaudhari, Azra Fatima, Soghra Bahmanpour i in. "Ascorbic Acid-Induced Cardiac Differentiation of Murine Pluripotent Stem Cells: Transcriptional Profiling and Effect of a Small Molecule Synergist of Wnt/β-Catenin Signaling Pathway". Cellular Physiology and Biochemistry 36, nr 2 (2015): 810–30. http://dx.doi.org/10.1159/000430140.
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Background: Reproducible and efficient differentiation of pluripotent stem cells (PSCs) to cardiomyocytes (CMs) is essential for their use in regenerative medicine, drug testing and disease modeling. The aim of this study was to evaluate the effect of some previously reported cardiogenic substances on cardiac differentiation of mouse PSCs. Methods: Differentiation was performed by embryoid body (EB)-based method using three different murine PSC lines. The differentiation efficiency was monitored by RT-qPCR, immunocytochemistry and flow cytometry, and the effect mechanistically evaluated by transcriptome analysis of treated EBs. Results: Among the five tested compounds (ascorbic acid, dorsomorphin, cyclic adenosine 3',5'-monophosphate, cardiogenol C, cyclosporin A) only ascorbic acid (AA) exerted a strong and reproducible cardiogenic effect in CGR8 cells which was less consistent in other two PSC lines. AA induced only minor changes in transcriptome of CGR8 cells after administration during the initial two days of differentiation. Cardiospecific genes and transcripts involved in angiogenesis, erythropoiesis and hematopoiesis were up-regulated on day 5 but not on days 2 or 3 of differentiation. The cardiac differentiation efficiency was improved when QS11, a small-molecule synergist of Wnt/β-catenin signaling pathway, was added to cultures after AA-treatment. Conclusion: This study demonstrates that only minor transcriptional changes are sufficient for enhancement of cardiogenesis of murine PSCs by AA and that AA and QS11 exhibit synergistic effects and enhance the efficiency of CM differentiation of murine PSCs.
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Bhatia, Vandanajay, Cristiana Rastellini, Song Han, Judith F. Aronson, George H. Greeley i Miriam Falzon. "Acinar cell-specific knockout of the PTHrP gene decreases the proinflammatory and profibrotic responses in pancreatitis". American Journal of Physiology-Gastrointestinal and Liver Physiology 307, nr 5 (1.09.2014): G533—G549. http://dx.doi.org/10.1152/ajpgi.00428.2013.
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Pancreatitis is a necroinflammatory disease with acute and chronic manifestations. Accumulated damage incurred during repeated bouts of acute pancreatitis (AP) can lead to chronic pancreatitis (CP). Pancreatic parathyroid hormone-related protein (PTHrP) levels are elevated in a mouse model of cerulein-induced AP. Here, we show elevated PTHrP levels in mouse models of pancreatitis induced by chronic cerulein administration and pancreatic duct ligation. Because acinar cells play a major role in the pathophysiology of pancreatitis, mice with acinar cell-specific targeted disruption of the Pthrp gene ( PTHrP Δacinar) were generated to assess the role of acinar cell-secreted PTHrP in pancreatitis. These mice were generated using Cre-LoxP technology and the acinar cell-specific elastase promoter. PTHrP Δacinar exerted protective effects in cerulein and pancreatic duct ligation models, evident as decreased edema, histological damage, amylase secretion, pancreatic stellate cell (PSC) activation, and extracellular matrix deposition. Treating acinar cells in vitro with cerulein increased IL-6 expression and NF-κB activity; these effects were attenuated in PTHrP Δacinar cells, as were the cerulein- and carbachol-induced elevations in amylase secretion. The cerulein-induced upregulation of procollagen I expression was lost in PSCs from PTHrP Δacinar mice. PTHrP immunostaining was elevated in human CP sections. The cerulein-induced upregulation of IL-6 and ICAM-1 (human acinar cells) and procollagen I (human PSCs) was suppressed by pretreatment with the PTH1R antagonist, PTHrP ( 7 – 34 ). These findings establish PTHrP as a novel mediator of inflammation and fibrosis associated with CP. Acinar cell-secreted PTHrP modulates acinar cell function via its effects on proinflammatory cytokine release and functions via a paracrine pathway to activate PSCs.
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Miyoshi, Norikatsu, Jente M. Stel, Keiko Shioda, Na Qu, Junko Odajima, Shino Mitsunaga, Xiangfan Zhang i in. "Erasure of DNA methylation, genomic imprints, and epimutations in a primordial germ-cell model derived from mouse pluripotent stem cells". Proceedings of the National Academy of Sciences 113, nr 34 (2.08.2016): 9545–50. http://dx.doi.org/10.1073/pnas.1610259113.
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The genome-wide depletion of 5-methylcytosines (5meCs) caused by passive dilution through DNA synthesis without daughter strand methylation and active enzymatic processes resulting in replacement of 5meCs with unmethylated cytosines is a hallmark of primordial germ cells (PGCs). Although recent studies have shown that in vitro differentiation of pluripotent stem cells (PSCs) to PGC-like cells (PGCLCs) mimics the in vivo differentiation of epiblast cells to PGCs, how DNA methylation status of PGCLCs resembles the dynamics of 5meC erasure in embryonic PGCs remains controversial. Here, by differential detection of genome-wide 5meC and 5-hydroxymethylcytosine (5hmeC) distributions by deep sequencing, we show that PGCLCs derived from mouse PSCs recapitulated the process of genome-wide DNA demethylation in embryonic PGCs, including significant demethylation of imprint control regions (ICRs) associated with increased mRNA expression of the corresponding imprinted genes. Although 5hmeCs were also significantly diminished in PGCLCs, they retained greater amounts of 5hmeCs than intragonadal PGCs. The genomes of both PGCLCs and PGCs selectively retained both 5meCs and 5hmeCs at a small number of repeat sequences such as GSAT_MM, of which the significant retention of bisulfite-resistant cytosines was corroborated by reanalysis of previously published whole-genome bisulfite sequencing data for intragonadal PGCs. PSCs harboring abnormal hypermethylation at ICRs of the Dlk1-Gtl2-Dio3 imprinting cluster diminished these 5meCs upon differentiation to PGCLCs, resulting in transcriptional reactivation of the Gtl2 gene. These observations support the usefulness of PGCLCs in studying the germline epigenetic erasure including imprinted genes, epimutations, and erasure-resistant loci, which may be involved in transgenerational epigenetic inheritance.
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Sherman, Mara H. "Abstract SY23-03: Mesenchymal lineage heterogeneity as a determinant of matrix composition and tumor progression". Cancer Research 82, nr 12_Supplement (15.06.2022): SY23–03—SY23–03. http://dx.doi.org/10.1158/1538-7445.am2022-sy23-03.
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Abstract The mechanisms underlying evolution of tumor-associated stroma remain poorly understood. In solid tumors featuring a prominent stromal reaction, an improved understanding of the functions and origins of abundant stromal cell types may facilitate the development of new and effective therapies. Pancreatic ductal adenocarcinoma (PDAC) is the quintessence of a fibro-inflammatory malignancy, with 50-90% of tumor volume occupied by a dense, desmoplastic stroma. Cancer-associated fibroblasts (CAFs) are the key cell type which drives the stromal reaction in PDAC, and recent reports suggest that stromal CAFs represent a heterogeneous population of cells from diverse origins, potentially including cell types which support and others which suppress tumor growth. Pancreatic stellate cells (PSCs) are lipid-storing cells in healthy pancreas which can transdifferentiate to an activated CAF phenotype. PSCs have been suggested as the predominant source of fibroblasts in the PDAC tumor microenvironment. However, proper lineage tracing studies have never been performed, such that the relative contribution and specific functions of PSCs in the tumor microenvironment are unknown. We have developed a novel mouse model to track PSC differentiation and function during pancreatic tumor progression in vivo. This model revealed that PSC-derived CAFs in fact give rise to a numerically minor but functionally significant subset of PDAC CAFs, and may represent a viable therapeutic target. We adapted our mouse model to enable targeted ablation of PSCs and derivative CAFs within their host tissue for the first time. Functional studies in this model reveal non-redundant functions for PSC-derived CAFs in shaping the PDAC microenvironment, and highlight the significance of mesenchymal lineage heterogeneity for pancreatic tumorigenesis. Further, we find that tumor genotype with respect to p53 status dictates stromal evolutionary routes with respect to cell of origin, a finding that may extend to additional cancer cell-intrinsic genomic alterations. Together, these findings shed light on mechanisms that shape the tumor microenvironment during pancreatic cancer progression, and may hold relevance in additional solid tumor types. Citation Format: Mara H. Sherman. Mesenchymal lineage heterogeneity as a determinant of matrix composition and tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY23-03.
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Su, Yue, Ling Wang, Zhiqiang Fan, Ying Liu, Jiaqi Zhu, Deborah Kaback, Julia Oudiz i in. "Establishment of Bovine-Induced Pluripotent Stem Cells". International Journal of Molecular Sciences 22, nr 19 (28.09.2021): 10489. http://dx.doi.org/10.3390/ijms221910489.
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Pluripotent stem cells (PSCs) have been successfully developed in many species. However, the establishment of bovine-induced pluripotent stem cells (biPSCs) has been challenging. Here we report the generation of biPSCs from bovine mesenchymal stem cells (bMSCs) by overexpression of lysine-specific demethylase 4A (KDM4A) and the other reprogramming factors OCT4, SOX2, KLF4, cMYC, LIN28, and NANOG (KdOSKMLN). These biPSCs exhibited silenced transgene expression at passage 10, and had prolonged self-renewal capacity for over 70 passages. The biPSCs have flat, primed-like PSC colony morphology in combined media of knockout serum replacement (KSR) and mTeSR, but switched to dome-shaped, naïve-like PSC colony morphology in mTeSR medium and 2i/LIF with single cell colonization capacity. These cells have comparable proliferation rate to the reported primed- or naïve-state human PSCs, with three-germ layer differentiation capacity and normal karyotype. Transcriptome analysis revealed a high similarity of biPSCs to reported bovine embryonic stem cells (ESCs) and embryos. The naïve-like biPSCs can be incorporated into mouse embryos, with the extended capacity of integration into extra-embryonic tissues. Finally, at least 24.5% cloning efficiency could be obtained in nuclear transfer (NT) experiment using late passage biPSCs as nuclear donors. Our report represents a significant advance in the establishment of bovine PSCs.
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Sakai, Yoshitake, Tomonori Nakamura, Ikuhiro Okamoto, Sayuri Gyobu-Motani, Hiroshi Ohta, Yukihiro Yabuta, Tomoyuki Tsukiyama i in. "Induction of the germ cell fate from pluripotent stem cells in cynomolgus monkeys†". Biology of Reproduction 102, nr 3 (13.11.2019): 620–38. http://dx.doi.org/10.1093/biolre/ioz205.
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Abstract In vitro reconstitution of germ-cell development from pluripotent stem cells (PSCs) has created key opportunities to explore the fundamental mechanisms underlying germ-cell development, particularly in mice and humans. Importantly, such investigations have clarified critical species differences in the mechanisms regulating mouse and human germ-cell development, highlighting the necessity of establishing an in vitro germ-cell development system in other mammals, such as non-human primates. Here, we show that multiple lines of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in cynomolgus monkeys (Macaca fascicularis; cy) can be maintained stably in an undifferentiated state under a defined condition with an inhibitor for WNT signaling, and such PSCs are induced efficiently into primordial germ cell-like cells (PGCLCs) bearing a transcriptome similar to early cyPGCs. Interestingly, the induction kinetics of cyPGCLCs from cyPSCs is faster than that of human (h) PGCLCs from hPSCs, and while the transcriptome dynamics during cyPGCLC induction is relatively similar to that during hPGCLC induction, it is substantially divergent from that during mouse (m) PGCLC induction. Our findings delineate common as well as species-specific traits for PGC specification, creating a foundation for parallel investigations into the mechanism for germ-cell development in mice, monkeys, and humans.
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Henríquez, Joaquín Araos, Judhell Manansala, Sara Pinto Teles, Muntadher Jihad, Eloise G. Lloyd, Amir Jassim, Richard J. Gilbertson i Giulia Biffi. "Abstract PR016: Aging modulates the tumor microenvironment of pancreatic cancer". Cancer Research 82, nr 22_Supplement (15.11.2022): PR016. http://dx.doi.org/10.1158/1538-7445.panca22-pr016.
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Abstract Pancreatic ductal adenocarcinoma (PDAC) has a very poor clinical outcome due to late diagnosis and resistance to therapies. Its epithelial compartment is surrounded by a cancer-associated fibroblast (CAF)-rich stroma that has been shown to be highly heterogeneous and promote therapy resistance and tumor growth. Importantly, the strongest risk factor for PDAC is age and half of patients are diagnosed when they are 75 years old or older. However, most pre-clinical studies of PDAC are performed on mouse models that are ~2-6 months old, which corresponds approximately to 20-30 human years. Thus, these models may not mimic the physiological state and molecular interactions between the tumor microenvironment (TME) and cancer cells of most PDAC cases. We hypothesize that the analysis of ageing models of PDAC will reveal new biology and targets for future therapeutic intervention. Our aim is to understand whether aging affects CAFs and immune cells and how this in turn impacts PDAC progression. To investigate this, we established new orthotopically-grafted organoid (OGO) aged mouse models of PDAC by injecting pancreatic cancer organoids derived from KPC (KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx1-Cre) mice into the pancreas of old (~18-month-old) C57BL/6J mice and compared these models to young (~3-month-old) OGO mice. We obtained tumor tissues for immunohistochemistry, flow cytometry and single-cell RNA-sequencing, plasma for cytokine arrays, and their tumor-free weight at endpoint. As a complementary strategy, we derived pancreatic stellate cells (PSCs), a precursor of CAFs, from old and young C57BL/6J mice and performed proliferation assays and transcriptomic analyses of PSCs cultured with PDAC organoid-conditioned media and of co-cultured KPC organoids and PSCs. We observed a significant weight loss and increasing concentrations of cytokines related to cancer cachexia in the plasma of aged PDAC mouse models in comparison to young mice. Moreover, we found that aging modulates the proliferative capacity, transcriptional signatures and gene interaction networks of CAFs in the PDAC TME. In particular, aging alters the composition of myofibroblastic CAF populations, while upregulating the expression of CAF inflammatory cytokines. Aging also reduced the abundance of macrophages and upregulated the expression of exhaustion-related genes in T cells. Moreover, we observed that old PSCs better support the proliferation of co-cultured PDAC organoids in growth factor-limiting conditions. This study suggests that aged mouse models of PDAC better recapitulate cachexia-related physiological changes observed in PDAC patients. Moreover, aging modulates aspects of the PDAC TME that are relevant for tumor progression, such as CAF composition and transcriptional signatures. We propose that these novel aged models could help identify new targets to improve the treatment of PDAC patients. Citation Format: Joaquín Araos Henríquez, Judhell Manansala, Sara Pinto Teles, Muntadher Jihad, Eloise G. Lloyd, Amir Jassim, Richard J. Gilbertson, Giulia Biffi. Aging modulates the tumor microenvironment of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR016.
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Gao, Xuxia, Yanna Cao, Wenli Yang, Chaojun Duan, Judith F. Aronson, Cristiana Rastellini, Celia Chao, Mark R. Hellmich i Tien C. Ko. "BMP2 inhibits TGF-β-induced pancreatic stellate cell activation and extracellular matrix formation". American Journal of Physiology-Gastrointestinal and Liver Physiology 304, nr 9 (1.05.2013): G804—G813. http://dx.doi.org/10.1152/ajpgi.00306.2012.
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Activation of pancreatic stellate cells (PSCs) by transforming growth factor (TGF)-β is the key step in the development of pancreatic fibrosis, a common pathological feature of chronic pancreatitis (CP). Bone morphogenetic proteins (BMPs), members of the TGF-β superfamily, have anti-fibrogenic functions, in contrast to TGF-β, in the kidney, lung, and liver. However, it is not known whether BMPs have an anti-fibrogenic role in the pancreas. The current study was designed to investigate the potential anti-fibrogenic role of BMPs in the pancreas using an in vivo CP model and an in vitro PSC model. CP was induced by repetitive intraperitoneal injections of cerulein in adult Swiss Webster mice. The control mice received saline injections. Compared with the control, cerulein injections induced a time-dependent increase in acinar injury and progression of fibrosis and a steady increase in inflammation. Cerulein injections also induced increases of the extracellular matrix (ECM) protein fibronectin and of α-smooth muscle actin (α-SMA)-positive stellate cells (PSCs). The mice receiving cerulein injections showed increased BMP2 protein levels and phosphorylated Smad1 levels up to 4 wk and then declined at 8 wk to similar levels as the control. In vitro, the isolated mouse and human PSCs were cultured and pretreated with BMP2 followed by TGF-β treatment. BMP2 pretreatment inhibited TGF-β-induced α-SMA, fibronectin, and collagen type Ia expression. Knocking down Smad1 with small-interfering RNA reversed the inhibitory effect of BMP2 on TGF-β-induced α-SMA and fibronectin expression. Thus, BMP2 opposes the fibrogenic function of TGF-β in PSCs through the Smad1 signaling pathway.
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Yang, Ying, Katsuyuki Adachi, Megan A. Sheridan, Andrei P. Alexenko, Danny J. Schust, Laura C. Schulz, Toshihiko Ezashi i R. Michael Roberts. "Heightened potency of human pluripotent stem cell lines created by transient BMP4 exposure". Proceedings of the National Academy of Sciences 112, nr 18 (13.04.2015): E2337—E2346. http://dx.doi.org/10.1073/pnas.1504778112.
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Human pluripotent stem cells (PSCs) show epiblast-type pluripotency that is maintained with ACTIVIN/FGF2 signaling. Here, we report the acquisition of a unique stem cell phenotype by both human ES cells (hESCs) and induced pluripotent stem cells (iPSCs) in response to transient (24–36 h) exposure to bone morphogenetic protein 4 (BMP4) plus inhibitors of ACTIVIN signaling (A83-01) and FGF2 (PD173074), followed by trypsin dissociation and recovery of colonies capable of growing on a gelatin substratum in standard medium for human PSCs at low but not high FGF2 concentrations. The self-renewing cell lines stain weakly for CDX2 and strongly for NANOG, can be propagated clonally on either Matrigel or gelatin, and are morphologically distinct from human PSC progenitors on either substratum but still meet standard in vitro criteria for pluripotency. They form well-differentiated teratomas in immune-compromised mice that secrete human chorionic gonadotropin (hCG) into the host mouse and include small areas of trophoblast-like cells. The cells have a distinct transcriptome profile from the human PSCs from which they were derived (including higher expression of NANOG, LEFTY1, and LEFTY2). In nonconditioned medium lacking FGF2, the colonies spontaneously differentiated along multiple lineages, including trophoblast. They responded to PD173074 in the absence of both FGF2 and BMP4 by conversion to trophoblast, and especially syncytiotrophoblast, whereas an A83-01/PD173074 combination favored increased expression of HLA-G, a marker of extravillous trophoblast. Together, these data suggest that the cell lines exhibit totipotent potential and that BMP4 can prime human PSCs to a self-renewing alternative state permissive for trophoblast development. The results may have implications for regulation of lineage decisions in the early embryo.
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Wang, Xiaoxiao, Yunlong Xiang, Yang Yu, Ran Wang, Yu Zhang, Qianhua Xu, Hao Sun i in. "Formative pluripotent stem cells show features of epiblast cells poised for gastrulation". Cell Research 31, nr 5 (19.02.2021): 526–41. http://dx.doi.org/10.1038/s41422-021-00477-x.
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AbstractThe pluripotency of mammalian early and late epiblast could be recapitulated by naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), respectively. However, these two states of pluripotency may not be sufficient to reflect the full complexity and developmental potency of the epiblast during mammalian early development. Here we report the establishment of self-renewing formative pluripotent stem cells (fPSCs) which manifest features of epiblast cells poised for gastrulation. fPSCs can be established from different mouse ESCs, pre-/early-gastrula epiblasts and induced PSCs. Similar to pre-/early-gastrula epiblasts, fPSCs show the transcriptomic features of formative pluripotency, which are distinct from naïve ESCs and primed EpiSCs. fPSCs show the unique epigenetic states of E6.5 epiblast, including the super-bivalency of a large set of developmental genes. Just like epiblast cells immediately before gastrulation, fPSCs can efficiently differentiate into three germ layers and primordial germ cells (PGCs) in vitro. Thus, fPSCs highlight the feasibility of using PSCs to explore the development of mammalian epiblast.
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Hodges, Craig A., Renée LeMaire-Adkins i Patricia A. Hunt. "Coordinating the segregation of sister chromatids during the first meiotic division: evidence for sexual dimorphism". Journal of Cell Science 114, nr 13 (1.07.2001): 2417–26. http://dx.doi.org/10.1242/jcs.114.13.2417.
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Errors during the first meiotic division are common in our species, but virtually all occur during female meiosis. The reason why oogenesis is more error prone than spermatogenesis remains unknown. Normal segregation of homologous chromosomes at the first meiotic division (MI) requires coordinated behavior of the sister chromatids of each homolog. Failure of sister kinetochores to act cooperatively at MI, or precocious sister chromatid segregation (PSCS), has been postulated to be a major contributor to human nondisjunction. To investigate the factors that influence PSCS we utilized the XO mouse, since the chromatids of the single X chromosome frequently segregate at MI, and the propensity for PSCS is influenced by genetic background. Our studies demonstrate that the strain-specific differences in PSCS are due to the actions of an autosomal trans-acting factor or factors. Since components of the synaptonemal complex are thought to play a role in centromere cohesion and kinetochore orientation, we evaluated the behavior of the X chromosome at prophase to determine if this factor influenced the propensity of the chromosome for self-synapsis. We were unable to directly correlate synaptic differences with subsequent segregation behavior. However, unexpectedly, we uncovered a sexual dimorphism that may partially explain sex-specific differences in the fidelity of meiotic chromosome segregation. Specifically, in the male remnants of the synaptonemal complex remain associated with the centromeres until anaphase of the second meiotic division (MII), whereas in the female, all traces of synaptonemal complex (SC) protein components are lost from the chromosomes before the onset of the first meiotic division. This finding suggests a sex-specific difference in the components used to correctly segregate chromosomes during meiosis, and may provide a reason for the high error frequency during female meiosis.
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Daimon, Atsushi, Hirofumi Morihara, Kiichiro Tomoda, Natsuko Morita, Yoshinori Koishi, Kazuyoshi Kanki, Masahide Ohmichi i Michio Asahi. "Intravenously Injected Pluripotent Stem Cell–derived Cells Form Fetomaternal Vasculature and Prevent Miscarriage in Mouse". Cell Transplantation 29 (1.01.2020): 096368972097045. http://dx.doi.org/10.1177/0963689720970456.
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Miscarriage is the most common complication of pregnancy, and about 1% of pregnant women suffer a recurrence. Using a widely used mouse miscarriage model, we previously showed that intravenous injection of bone marrow (BM)-derived endothelial progenitor cells (EPCs) may prevent miscarriage. However, preparing enough BM-derived EPCs to treat a patient might be problematic. Here, we demonstrated the generation of mouse pluripotent stem cells (PSCs), propagation of sufficient PSC-derived cells with endothelial potential (PSC-EPs), and intravenous injection of the PSC-EPs into the mouse miscarriage model. We found that the injection prevented miscarriage. Three-dimensional reconstruction images of the decidua after tissue cleaning revealed robust fetomaternal neovascularization induced by the PSC-EP injection. Additionally, the injected PSC-EPs directly formed spiral arteries. These findings suggest that intravenous injection of PSC-EPs could become a promising remedy for recurrent miscarriage.
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Yoshimura, Yasuhiro, Atsuhiro Taguchi, Shunsuke Tanigawa, Junji Yatsuda, Tomomi Kamba, Satoru Takahashi, Hidetake Kurihara, Masashi Mukoyama i Ryuichi Nishinakamura. "Manipulation of Nephron-Patterning Signals Enables Selective Induction of Podocytes from Human Pluripotent Stem Cells". Journal of the American Society of Nephrology 30, nr 2 (11.01.2019): 304–21. http://dx.doi.org/10.1681/asn.2018070747.
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BackgroundPrevious research has elucidated the signals required to induce nephron progenitor cells (NPCs) from pluripotent stem cells (PSCs), enabling the generation of kidney organoids. However, selectively controlling differentiation of NPCs to podocytes has been a challenge.MethodsWe investigated the effects of various growth factors in cultured mouse embryonic NPCs during three distinct steps of nephron patterning: from NPC to pretubular aggregate, from the latter to epithelial renal vesicle (RV), and from RV to podocyte. We then applied the findings to human PSC-derived NPCs to establish a method for selective induction of human podocytes.ResultsMouse NPC differentiation experiments revealed that phase-specific manipulation of Wnt and Tgf-β signaling is critical for podocyte differentiation. First, optimal timing and intensity of Wnt signaling were essential for mesenchymal-to-epithelial transition and podocyte differentiation. Then, inhibition of Tgf-β signaling supported domination of the RV proximal domain. Inhibition of Tgf-β signaling in the third phase enriched the podocyte fraction by suppressing development of other nephron lineages. The resultant protocol enabled successful induction of human podocytes from PSCs with >90% purity. The induced podocytes exhibited global gene expression signatures comparable to those of adult human podocytes, had podocyte morphologic features (including foot process–like and slit diaphragm–like structures), and showed functional responsiveness to drug-induced injury.ConclusionsElucidation of signals that induce podocytes during the nephron-patterning process enabled us to establish a highly efficient method for selective induction of human podocytes from PSCs. These PSC-derived podocytes show molecular, morphologic, and functional characteristics of podocytes, and offer a new resource for disease modeling and nephrotoxicity testing.
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Kime, Cody, Masayo Sakaki-Yumoto, Leeanne Goodrich, Yohei Hayashi, Salma Sami, Rik Derynck, Michio Asahi, Barbara Panning, Shinya Yamanaka i Kiichiro Tomoda. "Autotaxin-mediated lipid signaling intersects with LIF and BMP signaling to promote the naive pluripotency transcription factor program". Proceedings of the National Academy of Sciences 113, nr 44 (13.10.2016): 12478–83. http://dx.doi.org/10.1073/pnas.1608564113.
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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.
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Xu, Zhuojin, Aaron M. Robitaille, Jason D. Berndt, Kathryn C. Davidson, Karin A. Fischer, Julie Mathieu, Jennifer C. Potter, Hannele Ruohola-Baker i Randall T. Moon. "Wnt/β-catenin signaling promotes self-renewal and inhibits the primed state transition in naïve human embryonic stem cells". Proceedings of the National Academy of Sciences 113, nr 42 (3.10.2016): E6382—E6390. http://dx.doi.org/10.1073/pnas.1613849113.
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In both mice and humans, pluripotent stem cells (PSCs) exist in at least two distinct states of pluripotency, known as the naïve and primed states. Our understanding of the intrinsic and extrinsic factors that enable PSCs to self-renew and to transition between different pluripotent states is important for understanding early development. In mouse embryonic stem cells (mESCs), Wnt proteins stimulate mESC self-renewal and support the naïve state. In human embryonic stem cells (hESCs), Wnt/β-catenin signaling is active in naïve-state hESCs and is reduced or absent in primed-state hESCs. However, the role of Wnt/β-catenin signaling in naïve hESCs remains largely unknown. Here, we demonstrate that inhibition of the secretion of Wnts or inhibition of the stabilization of β-catenin in naïve hESCs reduces cell proliferation and colony formation. Moreover, we show that addition of recombinant Wnt3a partially rescues cell proliferation in naïve hESCs caused by inhibition of Wnt secretion. Notably, inhibition of Wnt/β-catenin signaling in naïve hESCs did not cause differentiation. Instead, it induced primed hESC-like proteomic and metabolic profiles. Thus, our results suggest that naïve hESCs secrete Wnts that activate autocrine or paracrine Wnt/β-catenin signaling to promote efficient self-renewal and inhibit the transition to the primed state.
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Langer, Ellen M., Isabel A. English, Vidhi Shah, Kevin MacPherson, Kayleigh M. Kresse, Brittany L. Allen-Petersen, Colin J. Daniel, Mara H. Sherman, Andrew Adey i Rosalie C. Sears. "Abstract PO-113: The prolyl isomerase PIN1 plays a critical role in fibroblast differentiation states to support pancreatic cancer". Cancer Research 81, nr 22_Supplement (15.11.2021): PO—113—PO—113. http://dx.doi.org/10.1158/1538-7445.panca21-po-113.
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Abstract PIN1 is a phosphorylation-directed prolyl isomerase that alters the conformation and, therefore, the function of many proteins. PIN1 overexpression in cancer contributes to cancer cell-intrinsic phenotypes including cellular proliferation and migration. While its pro-tumor functions have generated interest in therapeutic targeting of PIN1 for cancer treatment, the effects of PIN1 inhibition on tumor-associated stromal phenotypes have not yet been studied. We assessed pancreatic cancer xenografts and genetically engineered p48-Cre; LSL-KrasG12D; p53R172H (KPC) mice that were treated with small molecule PIN1 inhibitors or crossed into a full body PIN1 knockout (Pin1−/−), and found that PIN1 inhibition or loss decreased tumor growth and extended overall survival. To interrogate a direct role for PIN1 in the stroma, we orthotopically injected a KPC cell line into syngeneic Pin1+/+ or Pin1−/− hosts and found dramatic reduction of tumor cell growth in Pin1−/− hosts. Further analysis of the Pin1−/− tumor microenvironment revealed decreased expression of alpha-SMA, a marker of myofibroblastic cancer associated fibroblasts (myCAFs), as well as decreased ECM deposition and/or organization. Pancreatic stellate cells (PSCs) activated in the tumor microenvironment play a major role in the deposition of ECM and secrete growth factors to support tumor cell proliferation and survival. We, therefore, interrogated the role of PIN1 in PSCs. We found that loss of PIN1 in PSCs inhibits TGF-beta-induced stellate cell activation into a myofibroblast phenotype. Single cell ATAC-seq analysis demonstrated that a subset of TGF-beta responsive changes to chromatin accessibility are impaired in the absence of PIN1, and suggests that specific transcription factor families may play a role in the PIN1-dependent response to TGF-beta. Further analysis of PSCs or CAFs with PIN1 loss indicated that, at baseline, these cells express gene programs consistent with the recently described antigen presenting CAFs (apCAFs). Finally, in addition to changes in cellular state and plasticity, we found that loss of PIN1 alters PSC secretion of paracrine factors that support oncogenic phenotypes. For example, PSCs with loss of PIN1 have reduced expression of HGF and increased expression of VEGF, resulting in altered cancer cell and vascular phenotypes. This work establishes a role for PIN1 in regulating fibroblast function and suggests that targeting PIN1 in cancer will have a broad anti-tumor effect. Our ongoing work continues to use 2D co-cultures, heterotypic 3D bioprinted tissues, and in vivo mouse models to interrogate the precise mechanisms by which PIN1 controls fibroblast phenotypes and impact of these changes on tumor phenotypes and outcomes. Citation Format: Ellen M. Langer, Isabel A. English, Vidhi Shah, Kevin MacPherson, Kayleigh M. Kresse, Brittany L. Allen-Petersen, Colin J. Daniel, Mara H. Sherman, Andrew Adey, Rosalie C. Sears. The prolyl isomerase PIN1 plays a critical role in fibroblast differentiation states to support pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-113.
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Kim, Hantai, Young Sun Kim, Yeon Ju Kim, Jungho Ha, Siung Sung, Jeong Hun Jang, Sunho Park, Jangho Kim, Kyunghoon Kim i Yun-Hoon Choung. "Development of otic organoids and their current status". Organoid 3 (25.04.2023): e7. http://dx.doi.org/10.51335/organoid.2023.3.e7.
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The inner ear is responsible for both hearing and balance in the body, and since the initial development of otic (inner ear) organoids from mouse pluripotent stem cells (PSCs) in 2013, significant advances have been made in this field. Bone morphogenetic proteins, fibroblast growth factors, and Wnt agonists, which are signaling molecules in the early development of the inner ear, can induce PSCs into the otic fate. In the inner ear, hair cells and the surrounding supporting cells are essential for proper function and structure. Recent advancements in otic organoid research have enabled the generation of cells that closely resemble these key components. The developed otic organoids contain both hair cell-like cells and supporting cells, which have been confirmed to have the intrinsic function of those cell types. Otic organoids have been used for disease modeling and are expected to be more widely applied in various areas of research on the inner ear. However, the otic organoids developed to date remain immature. Although they mimic hair cells, their properties resemble vestibular (balance) hair cells more closely than cochlear (auditory) hair cells. The ultimate goal of research on the inner ear is hearing restoration and prevention; thus, it is essential to produce otic organoids that contain cochlear hair cells. In addition, the organ of Corti—a cell arrangement unique to the cochlea—has not yet been simulated. Along with a description of the current status of otic organoids, this review article will discuss future directions for otic organoids in inner ear research.