Stem cell therapies have emerged as a promising treatment strategy for various diseases characterized by ischemic injury such as ischemic stroke. Cell survival after transplantation remains a critical issue. We investigated the impact of oxidative stress, being typically present in ischemically challenged tissue, on human dental pulp stem cells (hDPSC) and human mesenchymal stem cells (hMSC). We used oxygen-glucose deprivation (OGD) to induce oxidative stress in hDPSC and hMSC. OGD-induced generation of O2•- or H2O2 enhanced autophagy by inducing the expression of activating molecule in BECN1-regulated autophagy protein 1 (Ambra1) and Beclin1 in both cell types. However, hDPSC and hMSC pre-conditioning using reactive oxygen species (ROS) scavengers significantly repressed the expression of Ambra1 and Beclin1 and inactivated autophagy. O2•- or H2O2 acted upstream of autophagy, and the mechanism was unidirectional. Furthermore, our findings revealed ROS-p38-Erk1/2 involvement. Pre-treatment with selective inhibitors of p38 and Erk1/2 pathways (SB202190 and PD98059) reversed OGD effects on the expression of Ambra1 and Beclin1, suggesting that these pathways induced oxidative stress-mediated autophagy. SIRT3 depletion was found to be associated with increased oxidative stress and activation of p38 and Erk1/2 MAPKs pathways. Global ROS inhibition by NAC or a combination of polyethylene glycol-superoxide dismutase (PEG-SOD) and polyethylene glycol-catalase (PEG-catalase) further confirmed that O2•- or H2O2 or a combination of both impacts stems cell viability by inducing autophagy. Furthermore, autophagy inhibition by 3-methyladenine (3-MA) significantly improved hDPSC viability. These findings contribute to a better understanding of post-transplantation hDPSC and hMSC death and may deduce strategies to minimize therapeutic cell loss under oxidative stress.

• Klíčová slova
• Ambra1, Beclin1, H2O2, O2•−, autophagy, human dental pulp stem cell, human mesenchymal stem cell, oxygen-glucose deprivation,
• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• apoptóza MeSH
• autofagie * MeSH
• beclin 1 metabolismus   farmakologie   MeSH
• glukosa metabolismus   MeSH
• kmenové buňky metabolismus   MeSH
• kyslík farmakologie   MeSH
• lidé MeSH
• oxidační stres MeSH
• peroxid vodíku * farmakologie   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• AMBRA1 protein, human MeSH Prohlížeč
• beclin 1 MeSH
• glukosa MeSH
• kyslík MeSH
• peroxid vodíku * MeSH
• reaktivní formy kyslíku MeSH

The mechanisms of obesity and type 2 diabetes (T2D)-associated impaired fracture healing are poorly studied. In a murine model of T2D reflecting both hyperinsulinemia induced by high-fat diet and insulinopenia induced by treatment with streptozotocin, we examined bone healing in a tibia cortical bone defect. A delayed bone healing was observed during hyperinsulinemia as newly formed bone was reduced by -28.4 ± 7.7% and was associated with accumulation of marrow adipocytes at the defect site +124.06 ± 38.71%, and increased density of SCA1+ (+74.99 ± 29.19%) but not Runx2+ osteoprogenitor cells. We also observed increased in reactive oxygen species production (+101.82 ± 33.05%), senescence gene signature (≈106.66 ± 34.03%), and LAMIN B1- senescent cell density (+225.18 ± 43.15%), suggesting accelerated senescence phenotype. During insulinopenia, a more pronounced delayed bone healing was observed with decreased newly formed bone to -34.9 ± 6.2% which was inversely correlated with glucose levels (R2 = 0.48, P < .004) and callus adipose tissue area (R2 = .3711, P < .01). Finally, to investigate the relevance to human physiology, we observed that sera from obese and T2D subjects had disease state-specific inhibitory effects on osteoblast-related gene signatures in human bone marrow stromal cells which resulted in inhibition of osteoblast and enhanced adipocyte differentiation. Our data demonstrate that T2D exerts negative effects on bone healing through inhibition of osteoblast differentiation of skeletal stem cells and induction of accelerated bone senescence and that the hyperglycemia per se and not just insulin levels is detrimental for bone healing.

• Klíčová slova
• bone healing, insulin-resistance, insulinopenia, senescence, type 2 diabetes,
• MeSH
• diabetes mellitus 2. typu * komplikace   MeSH
• fraktury kostí * MeSH
• hojení fraktur MeSH
• hyperinzulinismus * MeSH
• kmenové buňky MeSH
• kostní svalek MeSH
• lidé MeSH
• myši MeSH
• obezita komplikace   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

DNA damage repair (DDR) is a safeguard for genome integrity maintenance. Increasing DDR efficiency could increase the yield of induced pluripotent stem cells (iPSC) upon reprogramming from somatic cells. The epigenetic mechanisms governing DDR during iPSC reprogramming are not completely understood. Our goal was to evaluate the splicing isoforms of histone variant macroH2A1, macroH2A1.1, and macroH2A1.2, as potential regulators of DDR during iPSC reprogramming. GFP-Trap one-step isolation of mtagGFP-macroH2A1.1 or mtagGFP-macroH2A1.2 fusion proteins from overexpressing human cell lines, followed by liquid chromatography-tandem mass spectrometry analysis, uncovered macroH2A1.1 exclusive interaction with Poly-ADP Ribose Polymerase 1 (PARP1) and X-ray cross-complementing protein 1 (XRCC1). MacroH2A1.1 overexpression in U2OS-GFP reporter cells enhanced specifically nonhomologous end joining (NHEJ) repair pathway, while macroH2A1.1 knock-out (KO) mice showed an impaired DDR capacity. The exclusive interaction of macroH2A1.1, but not macroH2A1.2, with PARP1/XRCC1, was confirmed in human umbilical vein endothelial cells (HUVEC) undergoing reprogramming into iPSC through episomal vectors. In HUVEC, macroH2A1.1 overexpression activated transcriptional programs that enhanced DDR and reprogramming. Consistently, macroH2A1.1 but not macroH2A1.2 overexpression improved iPSC reprogramming. We propose the macroH2A1 splicing isoform macroH2A1.1 as a promising epigenetic target to improve iPSC genome stability and therapeutic potential.

• Klíčová slova
• DNA damage, cell reprogramming, induced pluripotent stem cells, macroH2A1.1,
• MeSH
• DNA MeSH
• endoteliální buňky metabolismus   MeSH
• histony * metabolismus   MeSH
• indukované pluripotentní kmenové buňky * metabolismus   MeSH
• lidé MeSH
• myši MeSH
• oprava DNA MeSH
• protein XRCC1 genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA MeSH
• histony * MeSH
• MACROH2A1 protein, human MeSH Prohlížeč
• protein XRCC1 MeSH
• XRCC1 protein, human MeSH Prohlížeč

MicroRNAs (miRNAs), a class of small, noncoding RNA molecules represent important regulators of gene expression. Recent reports have implicated their role in the cell specification process acting as "fine-tuners" to ensure the precise gene expression at the specific stage of cell differentiation. Here, we used retinal organoids differentiated from human pluripotent stem cells (hPSCs) as a model to closely investigate the role of a sensory organ-specific and evolutionary conserved miR-183/96/182 cluster. Using a miRNA tough decoy approach, we inhibited the miR-183/96/182 cluster in hPSCs. Inhibition of the miRNA cluster resulted in an increased expansion of neuroepithelium leading to abnormal "bulged" neural retina in organoids, associated with upregulation of neural-specific and retinal-specific genes. Importantly, we identified PAX6, a well-known essential gene in neuroectoderm specification, as a target of the miR-183/96/182 cluster members. Taken together, the miR-183/96/182 cluster not only represents an important regulator of PAX6 expression, but it also plays a crucial role in retinal tissue morphogenesis.

• Klíčová slova
• microRNAs, organoids, pluripotent stem cells, retina,
• Publikační typ
• časopisecké články MeSH

Three-dimensional (3D) retinal organoids, in vitro tissue structures derived from self-organizing cultures of differentiating human embryonic stem cells or induced pluripotent stem cells, could recapitulate some aspects of the cytoarchitectural structure and function of the retina in vivo. 3D retinal organoids display huge potential for the investigation of the pathogenesis of monogenic hereditary eye diseases that are related to the malfunction or degeneration of photoreceptors or retinal ganglion cells by providing an effective in vitro tool with multiple applications. In combination with recent genome editing tools, 3D retinal organoids could also represent a reliable and renewable source of transplantable cells for personalized therapies. In this review, we describe the recent advances in human pluripotent stem cells-derived retinal organoids, determination of their histoarchitecture, complexity, and maturity. We also discuss their application as a means to decipher the pathogenesis of retinal diseases, as well as the main drawbacks and challenges. Stem Cells 2019;37:1496-1504.

• Klíčová slova
• 3D organoids, Disease modeling, Induced pluripotent stem cells, Retinitis pigmentosa,
• MeSH
• editace genu metody   MeSH
• indukované pluripotentní kmenové buňky cytologie   transplantace   MeSH
• nemoci retiny patologie   MeSH
• organoidy cytologie   ultrastruktura   MeSH
• retina patologie   ultrastruktura   MeSH
• tyčinky retiny cytologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Transgenic mice expressing green fluorescent protein (GFP) are useful in transplantation experiments. When we used ubiquitin-GFP (UBC-GFP) transgenic mice to study the availability of niches for transplanted hematopoietic stem and progenitor cells, the results were strikingly different from the corresponding experiments that used congenic mice polymorphic in the CD45 antigen. Analysis of these unexpected results revealed that the hematopoiesis of UBC-GFP mice was outcompeted by the hematopoiesis of wild-type (WT) mice. Importantly, UBC-GFP mice engrafted the transplanted bone marrow of WT mice without conditioning. There was a significant bias toward lymphopoiesis in the WT branch of chimeric UBC-GFP/WT hematopoiesis. A fraction of immature Sca-1+ cells in the spleen of UBC-GFP mice expressed GFP at a very high level. The chimeric hematopoiesis was stable in the long term and also after transplantation to secondary recipient mice. The article thus identifies a specific defect in the hematopoiesis of UBC-GFP transgenic mice that compromises the lymphoid-primed hematopoietic stem cells in the bone marrow and spleen. Stem Cells 2018;36:1237-1248.

• Klíčová slova
• Bone marrow transplantation, Cell competition, Extramedullary hematopoiesis, Green fluorescent protein, Hematopoiesis, Lymphopoiesis, Stem cell niche, Stem cells, Transgenic mouse,
• MeSH
• chiméra MeSH
• hematopoetické kmenové buňky metabolismus   MeSH
• hematopoéza MeSH
• kostní dřeň metabolismus   MeSH
• lymfocyty metabolismus   MeSH
• lymfopoéza MeSH
• myši inbrední C57BL MeSH
• myši transgenní MeSH
• slezina metabolismus   MeSH
• splenektomie MeSH
• thymus metabolismus   MeSH
• transplantace hematopoetických kmenových buněk * MeSH
• ubikvitin metabolismus   MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ubikvitin MeSH
• zelené fluorescenční proteiny MeSH

Hereditary retinal dystrophies, specifically retinitis pigmentosa (RP) are clinically and genetically heterogeneous diseases affecting primarily retinal cells and retinal pigment epithelial cells with blindness as a final outcome. Understanding the pathogenicity behind these diseases has been largely precluded by the unavailability of affected tissue from patients, large genetic heterogeneity and animal models that do not faithfully represent some human diseases. A landmark discovery of human induced pluripotent stem cells (hiPSCs) permitted the derivation of patient-specific cells. These cells have unlimited self-renewing capacity and the ability to differentiate into RP-affected cell types, allowing the studies of disease mechanism, drug discovery, and cell replacement therapies, both as individual cell types and organoid cultures. Together with precise genome editing, the patient specific hiPSC technology offers novel strategies for targeting the pathogenic mutations and design therapies toward retinal dystrophies. This study summarizes current hiPSC-based RP models and highlights key achievements and challenges of these cellular models, as well as questions that still remain unanswered. Stem Cells 2018;36:474-481.

• Klíčová slova
• Differentiation, Gene targeting, Induced pluripotent stem, Induced pluripotent stem cells, Retina, Retinal photoreceptors, Retinal pigmented epithelium,
• MeSH
• autologní štěp MeSH
• buněčná diferenciace * MeSH
• editace genu * MeSH
• genom lidský * MeSH
• indukované pluripotentní kmenové buňky metabolismus   patologie   MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• retinopathia pigmentosa * genetika   metabolismus   patologie   terapie   MeSH
• transplantace kmenových buněk * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Hematopoietic stem cells derived from pluripotent stem cells could be used as an alternative to bone marrow transplants. Deriving these has been a long-term goal for researchers. However, the success of these efforts has been limited with the cells produced able to engraft in the bone marrow of recipient animals only in very low numbers. There is evidence that defects in the migratory and homing capacity of the cells are due to mis-regulation of miRNA expression and are responsible for their failure to engraft. We compared the miRNA expression profile of hematopoietic progenitors derived from pluripotent stem cells to those derived from bone marrow and found that numerous miRNAs are too highly expressed in hematopoietic progenitors derived from pluripotent stem cells, and that most of these are inhibitors of epithelial-mesenchymal transition or metastasis (including miR-200b, miR-200c, miR-205, miR-148a, and miR-424). We hypothesize that the high expression of these factors, which promote an adherent phenotype, may be causing the defect in hematopoietic differentiation. However, inhibiting these miRNAs, individually or in multiplex, was insufficient to improve hematopoietic differentiation in vitro, suggesting that other miRNAs and/or genes may be involved in this process. Stem Cells 2018;36:55-64.

• Klíčová slova
• Epithelial-mesenchymal transition, Hematopoietic differentiation, Human embryonic stem cells, miRNAs,
• MeSH
• buněčná diferenciace MeSH
• down regulace MeSH
• epitelo-mezenchymální tranzice genetika   MeSH
• hematopoetické kmenové buňky metabolismus   MeSH
• lidé MeSH
• mikro RNA genetika   MeSH
• pluripotentní kmenové buňky metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mikro RNA MeSH

Human pluripotent stem cells (hPSC) require signaling provided by fibroblast growth factor (FGF) receptors. This can be initiated by the recombinant FGF2 ligand supplied exogenously, but hPSC further support their niche by secretion of endogenous FGF2. In this study, we describe a role of tyrosine kinase expressed in hepatocellular carcinoma (TEC) kinase in this process. We show that TEC-mediated FGF2 secretion is essential for hPSC self-renewal, and its lack mediates specific differentiation. Following both short hairpin RNA- and small interfering RNA-mediated TEC knockdown, hPSC secretes less FGF2. This impairs hPSC proliferation that can be rescued by increasing amounts of recombinant FGF2. TEC downregulation further leads to a lower expression of the pluripotency markers, an improved priming towards neuroectodermal lineage, and a failure to develop cardiac mesoderm. Our data thus demonstrate that TEC is yet another regulator of FGF2-mediated hPSC pluripotency and differentiation. Stem Cells 2017;35:2050-2059.

• Klíčová slova
• Cardiac differentiation, Embryonic stem cells, Fibroblast growth factor, Fibroblast growth factor 2, Neural differentiation, Pluripotent stem cells, Tyrosine kinase expressed in hepatocellular carcinoma,
• MeSH
• biologické markery metabolismus   MeSH
• buněčné linie MeSH
• buněčný rodokmen * účinky léků   MeSH
• down regulace účinky léků   MeSH
• fibroblastový růstový faktor 2 metabolismus   MeSH
• lidé MeSH
• pluripotentní kmenové buňky cytologie   enzymologie   MeSH
• proliferace buněk účinky léků   MeSH
• rekombinantní proteiny farmakologie   MeSH
• tyrosinkinasy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• biologické markery MeSH
• fibroblastový růstový faktor 2 MeSH
• rekombinantní proteiny MeSH
• Tec protein-tyrosine kinase MeSH Prohlížeč
• tyrosinkinasy MeSH

The imitation switch nuclear ATPase Smarca5 (Snf2h) is one of the most conserved chromatin remodeling factors. It exists in a variety of oligosubunit complexes that move DNA with respect to the histone octamer to generate regularly spaced nucleosomal arrays. Smarca5 interacts with different accessory proteins and represents a molecular motor for DNA replication, repair, and transcription. We deleted Smarca5 at the onset of definitive hematopoiesis (Vav1-iCre) and observed that animals die during late fetal development due to anemia. Hematopoietic stem and progenitor cells accumulated but their maturation toward erythroid and myeloid lineages was inhibited. Proerythroblasts were dysplastic while basophilic erythroblasts were blocked in G2/M and depleted. Smarca5 deficiency led to increased p53 levels, its activation at two residues, one associated with DNA damage (S15Ph °s ) second with CBP/p300 (K376Ac ), and finally activation of the p53 targets. We also deleted Smarca5 in committed erythroid cells (Epor-iCre) and observed that animals were anemic postnatally. Furthermore, 4-hydroxytamoxifen-mediated deletion of Smarca5 in the ex vivo cultures confirmed its requirement for erythroid cell proliferation. Thus, Smarca5 plays indispensable roles during early hematopoiesis and erythropoiesis. Stem Cells 2017;35:1614-1623.

• Klíčová slova
• Cell cycle progression, Erythroid differentiation, Fetal liver erythropoiesis, Hematopoietic stem and progenitor cells, Hypoxia, Imitation switch, Smarca5, p53 pathway,
• MeSH
• adenosintrifosfatasy nedostatek   metabolismus   MeSH
• anemie patologie   MeSH
• buněčná diferenciace * MeSH
• buněčný cyklus MeSH
• chromozomální proteiny, nehistonové nedostatek   metabolismus   MeSH
• delece genu MeSH
• erytroidní buňky cytologie   MeSH
• erytropoéza MeSH
• genotyp MeSH
• hematopoetické kmenové buňky cytologie   metabolismus   MeSH
• hematopoéza MeSH
• messenger RNA genetika   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• nádorový supresorový protein p53 metabolismus   MeSH
• poškození DNA genetika   MeSH
• proliferace buněk MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• adenosintrifosfatasy MeSH
• chromozomální proteiny, nehistonové MeSH
• messenger RNA MeSH
• nádorový supresorový protein p53 MeSH
• Smarca5 protein, mouse MeSH Prohlížeč