Glial fibrillary acidic protein (GFAP) is a type-3 intermediate filament protein mainly expressed in astrocytes in the central nervous system. Mutations in GFAP cause Alexander disease (AxD), a rare and fatal neurological disorder. How exactly mutant GFAP eventually leads to white and gray matter deterioration in AxD remains unknown. GFAP is known to be expressed also in neural precursor cells in the developing brain. Here, we used AxD patient-derived induced pluripotent stem cells (iPSCs) to explore the impact of mutant GFAP during neurodifferentiation. Our results show that GFAP is already expressed in iPSCs. Moreover, we have found that mutations in GFAP can severely affect neural organoid development through altering lineage commitment in embryoid bodies. Together, these results support the notion that GFAP plays a role as an early modulator of neurodevelopment.

• MeSH
• Alexanderova nemoc genetika   patologie   MeSH
• buněčná diferenciace genetika   fyziologie   MeSH
• buněčný rodokmen * genetika   MeSH
• gliový fibrilární kyselý protein * genetika   metabolismus   MeSH
• indukované pluripotentní kmenové buňky metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mutace * genetika   MeSH
• organoidy * metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The formation of hematopoietic cells relies on the chromatin remodeling activities of ISWI ATPase SMARCA5 (SNF2H) and its complexes. The Smarca5 null and conditional alleles have been used to study its functions in embryonic and organ development in mice. These mouse model phenotypes vary from embryonic lethality of constitutive knockout to less severe phenotypes observed in tissue-specific Smarca5 deletions, e.g., in the hematopoietic system. Here we show that, in a gene dosage-dependent manner, the hypomorphic allele of SMARCA5 (S5tg) can rescue not only the developmental arrest in hematopoiesis in the hCD2iCre model but also the lethal phenotypes associated with constitutive Smarca5 deletion or Vav1iCre-driven conditional knockout in hematopoietic progenitor cells. Interestingly, the latter model also provided evidence for the role of SMARCA5 expression level in hematopoietic stem cells, as the Vav1iCre S5tg animals accumulate stem and progenitor cells. Furthermore, their hematopoietic stem cells exhibited impaired lymphoid lineage entry and differentiation. This observation contrasts with the myeloid lineage which is developing without significant disturbances. Our findings indicate that animals with low expression of SMARCA5 exhibit normal embryonic development with altered lymphoid entry within the hematopoietic stem cell compartment.

• MeSH
• adenosintrifosfatasy metabolismus   MeSH
• buněčná diferenciace genetika   MeSH
• hematopoetické kmenové buňky * metabolismus   MeSH
• hematopoéza * genetika   MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Developmental exposure to environmental factors has been linked to obesity risk later in life. Nuclear receptors are molecular sensors that play critical roles during development and, as such, are prime candidates to explain the developmental programming of disease risk by environmental chemicals. We have previously characterized the obesogen tributyltin (TBT), which activates the nuclear receptors peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor (RXR) to increase adiposity in mice exposed in utero. Mesenchymal stem cells (MSCs) from these mice are biased toward the adipose lineage at the expense of the osteoblast lineage, and MSCs exposed to TBT in vitro are shunted toward the adipose fate in a PPARγ-dependent fashion. To address where in the adipogenic cascade TBT acts, we developed an in vitro commitment assay that permitted us to distinguish early commitment to the adipose lineage from subsequent differentiation. TBT and RXR activators (rexinoids) had potent effects in committing MSCs to the adipose lineage, whereas the strong PPARγ activator rosiglitazone was inactive. We show that activation of RXR is sufficient for adipogenic commitment and that rexinoids act through RXR to alter the transcriptome in a manner favoring adipogenic commitment. RXR activation alters expression of enhancer of zeste homolog 2 (EZH2) and modifies genome-wide histone 3 lysine 27 trimethylation (H3K27me3) in promoting adipose commitment and programming subsequent differentiation. These data offer insights into the roles of RXR and EZH2 in MSC lineage specification and shed light on how endocrine-disrupting chemicals such as TBT can reprogram stem cell fate.

• MeSH
• adipogeneze účinky léků   genetika   fyziologie   MeSH
• buněčná diferenciace účinky léků   genetika   MeSH
• chromatin účinky léků   fyziologie   MeSH
• endokrinní disruptory farmakologie   MeSH
• epigeneze genetická účinky léků   MeSH
• exprese genu účinky léků   MeSH
• EZH2 protein genetika   MeSH
• genový knockdown veterinární   MeSH
• mezenchymální kmenové buňky cytologie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• obezita etiologie   MeSH
• PPAR gama fyziologie   MeSH
• retinoidní X receptory účinky léků   fyziologie   MeSH
• sekvenční analýza RNA veterinární   MeSH
• trialkylcínové sloučeniny farmakologie   MeSH
• tukové buňky cytologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

T cell memory relies on the generation of antigen-specific progenitors with stem-like properties. However, the identity of these progenitors has remained unclear, precluding a full understanding of the differentiation trajectories that underpin the heterogeneity of antigen-experienced T cells. We used a systematic approach guided by single-cell RNA-sequencing data to map the organizational structure of the human CD8+ memory T cell pool under physiological conditions. We identified two previously unrecognized subsets of clonally, epigenetically, functionally, phenotypically and transcriptionally distinct stem-like CD8+ memory T cells. Progenitors lacking the inhibitory receptors programmed death-1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) were committed to a functional lineage, whereas progenitors expressing PD-1 and TIGIT were committed to a dysfunctional, exhausted-like lineage. Collectively, these data reveal the existence of parallel differentiation programs in the human CD8+ memory T cell pool, with potentially broad implications for the development of immunotherapies and vaccines.

• MeSH
• biologické markery MeSH
• buněčná diferenciace imunologie   MeSH
• CD8-pozitivní T-lymfocyty imunologie   metabolismus   MeSH
• homeostáza telomer MeSH
• imunofenotypizace MeSH
• imunologická paměť * MeSH
• lidé MeSH
• lymfoidní progenitorové buňky cytologie   imunologie   metabolismus   MeSH
• myši MeSH
• stanovení celkové genové exprese MeSH
• T-lymfocyty - podskupiny imunologie   metabolismus   MeSH
• výpočetní biologie metody   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

During embryogenesis, the Activin/Nodal pathway promotes the mesendodermal lineage and inhibits neural fate. The molecular mechanisms underlying this role of the Activin/Nodal pathway are not clear. In this study, we report a role for protein tyrosine phosphatase 1B (PTP1B) in Activin-mediated early fate decisions during ESC differentiation and show that PTP1B acts as an effector of the Activin pathway to specify mesendodermal or neural fate. We found that the Activin/ALK4 pathway directly recruits PTP1B and stimulates its release from the endoplasmic reticulum through ALK4-mediated cleavage. Subsequently, PTP1B suppresses p-ERK1/2 signaling to inhibit neural specification and promote mesendodermal commitment. These findings suggest that a noncanonical Activin signaling pathway functions in lineage specification of mouse and human embryonic stem cells.

• MeSH
• aktivinové receptory typu I chemie   metabolismus   MeSH
• aktiviny metabolismus   MeSH
• benzamidy farmakologie   MeSH
• buněčná diferenciace * účinky léků   MeSH
• buněčný rodokmen účinky léků   MeSH
• dioxoly farmakologie   MeSH
• embryonální kmenové buňky cytologie   účinky léků   enzymologie   MeSH
• endoderm cytologie   účinky léků   metabolismus   MeSH
• extracelulárním signálem regulované MAP kinasy metabolismus   MeSH
• fosforylace účinky léků   MeSH
• lidé MeSH
• MAP kinasový signální systém účinky léků   MeSH
• mezoderm cytologie   účinky léků   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• myši MeSH
• neurony cytologie   účinky léků   metabolismus   MeSH
• pluripotentní kmenové buňky cytologie   účinky léků   metabolismus   MeSH
• protein Smad2 metabolismus   MeSH
• sekvence aminokyselin MeSH
• signální transdukce * účinky léků   MeSH
• tyrosinfosfatasa nereceptorového typu 1 metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

OBJECTIVE: The primary objective of this study was to identify and clone the first nonmammalian thrombopoietin (TPO), chicken TPO, and its receptor c-Mpl for the purpose of characterizing their activities both in vitro and in vivo. MATERIALS AND METHODS: Chicken TPO was cloned using the methods of reverse transcriptase polymerase chain reaction and rapid amplification of cDNA ends. Northern blotting and RNAse protection assays were employed to analyze the levels of RNA expression in a panel of tissues and cell lines. To study cell surface expression of c-Mpl, polyclonal antibodies were prepared against bacterially derived c-Mpl. Both baculovirus-derived recombinant TPO and retrovirally expressed TPO and c-Mpl were used for the in vivo experiments. RESULTS: Both chicken TPO and its receptor c-Mpl were identified and cloned. Expression of chicken TPO was restricted to only the liver and spleen, while c-mpl was expressed in the bone marrow, lung, and spleen. In vitro experiments with sorted multipotent chicken bone marrow-derived progenitors demonstrated that TPO plays a role in the commitment of these cells to the thrombocytic lineage. Furthermore, TPO in cooperation with stem cell factor also supports proliferation of multipotent progenitors. In experimental animals, the intravenous application of recombinant chicken TPO or overexpression of TPO and c-mpl via retroviral infection lead to erythroblastosis and thromboblastosis. CONCLUSION: The characterized chicken thrombopoietin and its receptor c-Mpl will be valuable tools to further study thrombocytic differentiation and hematopoietic stem cell development. Moreover, the introduced experimental model of the chicken bipotent thrombo-/erythropoietic-progenitor can be used to identify key regulators of cell fate determination.

• MeSH
• buněčný rodokmen MeSH
• Escherichia coli genetika   MeSH
• fibroblasty cytologie   MeSH
• financování organizované MeSH
• hematopoéza fyziologie   MeSH
• klonování DNA MeSH
• kur domácí MeSH
• kuřecí embryo MeSH
• lidé MeSH
• messenger RNA biosyntéza   MeSH
• molekulární sekvence - údaje MeSH
• myši MeSH
• receptory thrombopoetinu biosyntéza   fyziologie   genetika   MeSH
• rekombinantní proteiny genetika   MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• thrombopoetin biosyntéza   fyziologie   genetika   MeSH
• zvířata MeSH
• Check Tag
• kuřecí embryo MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH

Human multipotent neural stem cells could effectively be used for the treatment of a variety of neurological disorders. However, a defining signature of neural stem cell lines that would be expandable, non-tumorigenic, and differentiate into desirable neuronal/glial phenotype after in vivo grafting is not yet defined. Employing a mass spectrometry approach, based on selected reaction monitoring, we tested a panel of well-described culture conditions, and measured levels of protein markers routinely used to probe neural differentiation, i.e. POU5F1 (OCT4), SOX2, NES, DCX, TUBB3, MAP2, S100B, GFAP, GALC, and OLIG1. Our multiplexed assay enabled us to simultaneously identify the presence of pluripotent, multipotent, and lineage-committed neural cells, thus representing a powerful tool to optimize novel and highly specific propagation and differentiation protocols. The multiplexing capacity of this method permits the addition of other newly identified cell type-specific markers to further increase the specificity and quantitative accuracy in detecting targeted cell populations. Such an expandable assay may gain the advantage over traditional antibody-based assays, and represents a method of choice for quality control of neural stem cell lines intended for clinical use.

• MeSH
• biologické markery MeSH
• buněčná diferenciace * MeSH
• buněčné linie MeSH
• buněčný rodokmen genetika   MeSH
• hmotnostní spektrometrie MeSH
• imunohistochemie MeSH
• lidé MeSH
• nervové kmenové buňky cytologie   metabolismus   MeSH
• neuroglie MeSH
• neurony MeSH
• stanovení celkové genové exprese MeSH
• vývojová regulace genové exprese MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The maximal nuclear and cell body diameters of leukaemic agranular myeloblasts ("type 1" without azurophilic granules) were measured in bone marrow smears of patients suffering from acute myeloblastic leukaemia with minimal differentiation (M0 AML), without and with maturation (M1 and M2 AML), refractory anaemia with excess of myeloblasts (RAEB) and chronic phase of the myeloid leukaemia (CML) to provide more information on the cytoplasmic space estimate occupied by the nucleus. The largest size of the cytoplasmic space occupied by the nucleus in agranular myeloblasts was noted in M0 and M1 AML in comparison with M2 AML or RAEB, and especially with CML. Similarly, agranular myeloblasts with nuclear bodies occupying more than 90 per cent of the cell space were most frequent in M0 and M1 AML (> 50 %), less frequent in M2 and RAEB (~ 20 %) but very rare in CML (~ 5 %). According to electron microscopy, the very narrow cytoplasmic shell surrounding the nucleus in such myeloblasts did not possess any space for structural components characteristic of the granulocytic cell lineage. Thus, the basic morphology of these myeloblasts would correspond to morphological features of a less differentiated committed stem cell. It should be noted that such cells may be easily recognized in currently stained bone marrow or peripheral blood smears.

• MeSH
• akutní myeloidní leukemie * patologie   MeSH
• buněčná diferenciace * MeSH
• chronická myeloidní leukemie * patologie   MeSH
• cytoplazma * metabolismus   MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• myelodysplastické syndromy * patologie   MeSH
• prekurzorové buňky granulocytů patologie   MeSH
• retrospektivní studie MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

The epithelial-mesenchymal plasticity, in tight association with stemness, contributes to the mammary gland homeostasis, evolution of early neoplastic lesions and cancer dissemination. Focused on cell surfaceome, we used mouse models of pre-neoplastic mammary epithelial and cancer stem cells to reveal the connection between cell surface markers and distinct cell phenotypes. We mechanistically dissected the TGF-β family-driven regulation of Sca-1, one of the most commonly used adult stem cell markers. We further provided evidence that TGF-β disrupts the lineage commitment and promotes the accumulation of tumor-initiating cells in pre-neoplastic cells.

• MeSH
• ataxin-1 metabolismus   MeSH
• epitelo-mezenchymální tranzice genetika   MeSH
• epitelové buňky patologie   MeSH
• experimentální nádory mléčných žláz genetika   patologie   MeSH
• lidé MeSH
• mléčné žlázy zvířat patologie   MeSH
• myši MeSH
• nádorové buněčné linie transplantace   MeSH
• nádorové kmenové buňky patologie   MeSH
• nádory prsu genetika   patologie   MeSH
• plasticita buňky genetika   MeSH
• receptor erbB-2 genetika   MeSH
• regulace genové exprese u nádorů MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• signální transdukce genetika   MeSH
• transformující růstový faktor beta genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Emergency granulopoiesis is a critical process by which hematopoietic progenitors and stem cells facilitate enhanced granulocytic production during severe infections. However, the role of distinct multipotent progenitors (MPPs) at early stages of this process remains underexplored. Here, we investigated the contribution of MPPs to granulocytic production following lipopolysaccharide (LPS) administration in wild-type mice, simulating a bacterial infection. Transplantation assays demonstrated that LPS exposure reduces the engraftment capacity of lymphoid-biased MPP4 and enhances lymphoid production, rather than supporting myeloid lineage output. Further, single-cell RNA sequencing (scRNA-seq) of MPPs isolated from control and LPS-challenged mice revealed transcriptional reprogramming of nonlineage committed MPPs toward myeloid- and erythroid-biased progenitors. Notably, inflammatory progenitor populations emerged on activation of LPS-induced emergency granulopoiesis, displaying chromatin accessibility changes that align with a commitment to myeloid and erythroid fates. Pseudotime analysis elucidated cellular trajectories that suggest a developmental pathway where unbiased progenitors, present under nonstress conditions, transition toward myeloid and erythroid lineage outputs on LPS administration. In line with our functional MPP4 assessment, scRNA-seq suggested that lymphoid-biased progenitors do not transcriptionally rewire during early stages of emergency granulopoiesis. Collectively, our data highlight the critical role of specific MPP subsets in responding to LPS-induced inflammatory signals and underscore the dynamic adaptations that occur during granulocyte production in response to infection.

• MeSH
• genetická transkripce * účinky léků   MeSH
• granulocyty * metabolismus   cytologie   MeSH
• hematopoetické kmenové buňky * metabolismus   cytologie   MeSH
• hematopoéza * účinky léků   MeSH
• lipopolysacharidy * farmakologie   toxicita   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH