Obesity and type 2 diabetes are associated with disturbances in insulin-regulated glucose and lipid fluxes and severe comorbidities including cardiovascular disease and steatohepatitis. Whole body metabolism is regulated by lipid-storing white adipocytes as well as "brown" and "brite/beige" adipocytes that express thermogenic uncoupling protein 1 (UCP1) and secrete factors favorable to metabolic health. Implantation of brown fat into obese mice improves glucose tolerance, but translation to humans has been stymied by low abundance of primary human beige adipocytes. Here we apply methods to greatly expand human adipocyte progenitors from small samples of human subcutaneous adipose tissue and then disrupt the thermogenic suppressor gene NRIP1 by CRISPR. Ribonucleoprotein consisting of Cas9 and sgRNA delivered ex vivo are fully degraded by the human cells following high efficiency NRIP1 depletion without detectable off-target editing. Implantation of such CRISPR-enhanced human or mouse brown-like adipocytes into high fat diet fed mice decreases adiposity and liver triglycerides while enhancing glucose tolerance compared to implantation with unmodified adipocytes. These findings advance a therapeutic strategy to improve metabolic homeostasis through CRISPR-based genetic enhancement of human adipocytes without exposing the recipient to immunogenic Cas9 or delivery vectors.
- MeSH
- bílé tukové buňky metabolismus MeSH
- buněčná diferenciace MeSH
- buněčné kultury metody MeSH
- CRISPR-Cas systémy genetika MeSH
- dieta s vysokým obsahem tuků škodlivé účinky MeSH
- dospělé kmenové buňky fyziologie MeSH
- editace genu metody MeSH
- guide RNA, Kinetoplastida genetika MeSH
- hnědé tukové buňky metabolismus transplantace MeSH
- lidé MeSH
- metabolismus lipidů genetika MeSH
- modely nemocí na zvířatech MeSH
- myši MeSH
- nuclear receptor interacting protein 1 genetika metabolismus MeSH
- obezita komplikace metabolismus terapie MeSH
- podkožní tuk cytologie MeSH
- porucha glukózové tolerance etiologie metabolismus terapie MeSH
- termogeneze genetika MeSH
- ztučnělá játra etiologie metabolismus prevence a kontrola MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- 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
Adult stem cells are undifferentiated elements able to self-renew or differentiate to maintain tissue integrity. Within this context, stem cells are able to divide in a symmetric fashion, feature characterising all the somatic cells, or in an asymmetric way, which leads daughter cells to different fates. It is worth highlighting that cell polarity have a critical role in regulating stem cell asymmetric division and the proper control of cell division depends on different proteins involved in cell development, differentiation and maintenance of tissue homeostasis. Moreover, the interaction between cells and the extracellular matrix are crucial in influencing cell behavior, included in terms of mechanical properties as cytoskeleton plasticity and remodelling, and membrane tension. Finally, the activation of specific transcriptional program and epigenetic modifications contributes to cell fate determination, through modulation of cellular signalling cascades. It is well known that physical and mechanical stimuli are able to influence biological systems, and in this context, the effects of electromagnetic fields (EMFs) have already shown a considerable role, even though there is a lack of knowledge and much remains to be done around this topic. In this review, we summarize the historical background of EMFs applications and the main molecular mechanism involved in cellular remodelling, with particular attention to cytoskeleton elasticity and cell polarity, required for driving stem cell behavior.
The identification, enrichment and subsequent isolation of spermatogonial stem cells (SSCs) are integral to the success of SCC transplants between fertile donor and sterilized recipient males. In birds generally and particularly in chicken, SSC-specific has yet to be identified. The receptor for glial cell-derived neurotrophic factor (GDNF), i.e. GDNF family receptor alpha-1 (GFRα1), has been identified as a potential marker for different mouse spermatogonial subtypes. In the present study, we characterized the chicken cGFRα1 receptor and compared its predicted amino-acid sequence with mouse, rat and human GFRα1 proteins. Using specific polyclonal mouse anti-cGFRα1 serum, a total of 2.8% cells were recognized as cGFRα1-positive among isolated testicular cells recovered from sexually mature cockerels. The percentages of cGFRα1-positive testicular cells with haploid, diploid, tetraploid and SP DNA content were 1.6%, 2.5%, 39.3% and 76.8%, respectively. The presence of cGFRα1 protein on the surfaces of all cells of the seminiferous epithelium was confirmed by immunocytochemical and immunohistochemical analyses. Tissue specificity of cGFRα1 mRNA expression was significantly higher in adult testes compared to brain tissue which itself was several times higher than tissues prepared from the spleen, liver and heart. No expression was observed in muscular tissue. At last, we demonstrated the successful repopulation of sterilized recipient's testes with transplanted cGFRα1-positive donor testicular cells. Recipient males subsequently produced functional heterologous spermatozoa capable of fertilizing an ovum and obtaining chicks with donor cell genotypes.
- MeSH
- biologické markery MeSH
- dospělé kmenové buňky fyziologie MeSH
- genotyp MeSH
- klonování DNA MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- molekulární sekvence - údaje MeSH
- protilátky krev MeSH
- receptory GDNF genetika metabolismus MeSH
- regulace genové exprese fyziologie MeSH
- sekvence aminokyselin MeSH
- testis cytologie MeSH
- transplantace kmenových buněk veterinární MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The mouse Dach1 gene, involved in the development of the neocortex and the hippocampus, is expressed by neural stem cells (NSCs) during early neurogenesis, and its expression also continues in a subpopulation of cells in the dorsal part of the lateral ventricles (LV) of the adult mouse brain. In this study we aimed to elucidate the role of Dach1-expressing cells in adult neurogenesis/gliogenesis under physiological as well as post-ischemic conditions, employing transgenic mice in which the expression of green fluorescent protein (GFP) is controlled by the D6 promotor of the mouse Dach1 gene. A neurosphere-forming assay of GFP⁺ cells isolated from the dorsal part of the LV was carried out with subsequent differentiation in vitro. To elucidate the neurogenic/gliogenic potential of GFP⁺ cells in the dorsal part of the LV, in situ immunohistochemical/electrophysiological analyses of GFP⁺ cells in adult sham-operated brains (controls) and those after middle cerebral artery occlusion (MCAo) were performed. The GFP⁺ cells isolated from the dorsal part of the LV of controls formed neurospheres and differentiated solely into a glial phenotype, while those isolated after MCAo also gave rise to cells with the properties of neuronal precursors. In situ analyses revealed that GFP⁺ cells express the phenotype of adult NSCs or neuroblasts in controls as well as following ischemia. Following MCAo we found a significantly increased number of GFP⁺ cells expressing doublecortin as well as a number of GFP⁺ cells migrating through the rostral migratory stream into the olfactory bulb, where they probably differentiated into calretinin⁺ interneurons. Collectively, our results suggest the involvement of the mouse Dach1 gene in adult neurogenesis; cells expressing this gene exhibit the properties of adult NSCs or neuroblasts and respond to MCAo by enhanced neurogenesis.
- MeSH
- 4-aminopyridin farmakologie MeSH
- blokátory sodíkových kanálů farmakologie MeSH
- buněčná diferenciace fyziologie MeSH
- degenerace nervu etiologie patologie MeSH
- dospělé kmenové buňky fyziologie MeSH
- infarkt arteria cerebri media komplikace MeSH
- membránové potenciály účinky léků MeSH
- metoda terčíkového zámku MeSH
- modely nemocí na zvířatech MeSH
- myši inbrední C57BL MeSH
- myši transgenní MeSH
- myši MeSH
- neurogeneze fyziologie MeSH
- neurony metabolismus MeSH
- oční proteiny metabolismus MeSH
- počet buněk MeSH
- proteiny nervové tkáně metabolismus MeSH
- techniky in vitro MeSH
- tetraethylamonium farmakologie MeSH
- tetrodotoxin farmakologie MeSH
- ventriculi laterales patologie MeSH
- zelené fluorescenční proteiny genetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Kmenové buňky "dospělých" tkání ("adult tissue stem cells" - ATSC) představují unikátní populaci fenotypicky primitivních buněk, které hrají klíčovou roli ve fyziologické obměně tkání i v řadě patologických procesů. K základním vlastnostem ATCS patří schopnost proliferace, reprodukce a diferenciace v buněčné progenitory z nichž vznikají terminálně diferencované funkční buňky dané tkáně. Základním elementem hierarchicky uspořádaného systému ATSC a progenitorových buněk jsou totipotentní epiblastické buňky schopné diferenciace do buněk všech základních linií, tj. ektodermu, mezodermu i endodermu. Na dalších úrovních stojí pluripotentní liniově zaměřené buňky schopné diferenciace do všech buněčných typů jedné ze základních linií (jednoho zárodečného listu), tkáňově specifické progeni-torové buňky schopné diferenciace do jednoho nebo několika různých buněčných typů tvořících danou tkáň a přechodné dělící se buňky ("transit amplifying cells"), které vykazují vysokou proliferační aktivitu a které přecházejí do nedělících se terminálně diferencovaných buněk. Tkáňově specifické progenitorové buňky zajišťující fyziologickou obměnu sliznice GIT jsou v jednotlivých oddílech lokalizovány výhradně v určité oblasti sliznice. V jícnu jsou to interpapilární oblast bazální vrstvy dlaždicového epitelu a vývody mucinózních ezofageálních žlázek, v žaludeční sliznici oblast krčků žlázek a ve střevě baze krypt. Řada patologických procesů GIT je spojena s alteracemi ATCS a tkáňově specifických progenitorových buněk. U chronických zánětů jsou často rozšířeny úseky sliznice tvořené progenitorovými buňkami. Tento fenomén bývá doprovázen poruchou terminální diferenciace epitelových buněk. Změna tkáňového prostředí v terénu chronických zánětů může navodit alternativní diferenciaci progenitorových buněk. Typickými příklady jsou vznik specializovaného cylindrického epitelu Barrettova jícnu u refluxní ezofagitidy a intestinální metaplazie u chronické gastritidy. Komplexní alterace kmenových buněk na genové úrovni vedoucí k poruše regulačních mechanizmů buněčné proliferace, diferenciace a apoptózy jsou prvotní příčinou vzniku nádorových onemocnění.
Adult tissue stem cells (ATSC) are unique population of phenotypic primitive cells playing a key role in the physiologic turnover of cell of tissues and many pathologic processes as well. The basic feature of ATSC is the ability of proliferation, reproduction and diferentiation into cell progenitors, from which terminally differentiated fully functioning cells of a given tissue are generated. The basic elements of hierarchically arranged ATSC system are totipotent epiblastic cells able to diferentiate into cells of every basic lineages, ectoderm, mesoderm and endoderm as well. On further levels there are pluripotent lineages centered cells able to diferentiate into all cell types of one of the basic lineages (of one germinal leaf), tissue specific progenitor cells into diferentiation into one or more several cell types of a given tissue and transit amplifying cells with high proliferative activity and transfer into non-divided terminally diferentiated cells. Tissue specific progenitor cells managing the physiologic exchange of gastrointestinal mucosa are in every departments localised only in a specific region of mucosa. In esophagus there is a interpapillary part of basal stratum of squamous epithel and ducts of mucinous esophageal glands, in gastric mucosa the region of gland neck and the crypt bases in the gut. Several pathologic processes of gastrointestinal tract are joined to ATSC and tissue specific progenitor cells changes. In chronic inflammation there are the parts of mucosa from progenitor cell often dilated. This phenomenon is often accomplished by alteration of terminal diferentiation of progenitor cells. The changes of tissue enviroment in the field of inflammation can start an alternative diferentiation of progenitor cells. Typical examples are the specialised epithelium of Barretts esophagus in reflux esophagitis and intestinal metaplasia in chronic gastritis. Alteration of stem cells on the gene level leading to alteration of regulatory mechanisms of cell proliferation, diferentiation and apoptosis are the primary cause of tumorous tissue changes.
