Definitive endoderm (DE) is one of the three germ layers which during in vivo vertebrate development gives rise to a variety of organs including liver, lungs, thyroid and pancreas; consequently ...efficient in vitro initiation of stem cell differentiation to DE cells is a prerequisite for successful cellular specification to subsequent DE-derived cell types 1, 2. In this study we present a novel approach to rapidly and efficiently down regulate pluripotency genes during initiation of differentiation to DE cells by addition of dimethyl sulfoxide (DMSO) to Activin A-based culture medium and report its effects on the downstream differentiation to hepatocyte-like cells.
Human embryonic stem cells (hESC) were differentiated to DE using standard methods in medium supplemented with 100ng/ml of Activin A and compared to cultures where DE specification was additionally enhanced with different concentrations of DMSO. DE cells were subsequently primed to generate hepatic-like cells to investigate whether the addition of DMSO during formation of DE improved subsequent expression of hepatic markers. A combination of flow cytometry, real-time quantitative reverse PCR and immunofluorescence was applied throughout the differentiation process to monitor expression of pluripotency (POUF5/OCT4 & NANOG), definitive endoderm (SOX17, CXCR4 & GATA4) and hepatic (AFP & ALB) genes to generate differentiation stage-specific signatures.
Addition of DMSO to the Activin A-based medium during DE specification resulted in rapid down regulation of the pluripotency genes OCT4 and NANOG, accompanied by an increase expression of the DE genes SOX17, CXCR4 and GATA4. Importantly, the expression level of ALB in DMSO-treated cells was also higher than in cells which were differentiated to the DE stage via standard Activin A treatment.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Human primordial germ cells and mouse neonatal and adult germline stem cells are pluripotent and show similar properties to embryonic stem cells. Here we report the successful establishment of human ...adult germline stem cells derived from spermatogonial cells of adult human testis. Cellular and molecular characterization of these cells revealed many similarities to human embryonic stem cells, and the germline stem cells produced teratomas after transplantation into immunodeficient mice. The human adult germline stem cells differentiated into various types of somatic cells of all three germ layers when grown under conditions used to induce the differentiation of human embryonic stem cells. We conclude that the generation of human adult germline stem cells from testicular biopsies may provide simple and non-controversial access to individual cell-based therapy without the ethical and immunological problems associated with human embryonic stem cells.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract Background Reports of altered endogenous neurogenesis in people with Alzheimer’s disease (AD) and transgenic AD models have suggested that endogenous neurogenesis may be an important ...treatment target, but there is considerable discrepancy among studies. We examined endogenous neurogenesis and glia changes across the range of pathologic severity of AD in people with and without dementia to address this key question. Methods Endogenous neurogenesis and glia in the subventricular zone and dentate gyrus neurogenic niches were evaluated using single and double immunohistochemistry and a validated antibody selection for stage-specific and type-specific markers in autopsy tissue from a representative cohort of 28 participants in the Medical Research Council Cognitive Function and Ageing Study. Immunopositive cells were measured blinded to diagnosis using bright-field and fluorescent microscopy. Results The number of newly generated neurons significantly declined only in the dentate gyrus of patients with severe tau pathology. No other changes in other neurogenic markers were observed in either of the neurogenic niches. Alterations in astrocytes and microglia were also observed in the dentate gyrus across the different stages of tau pathology. No change in any of the markers was observed in individuals who died with dementia compared with individuals who did not die with dementia. Conclusions Alterations in endogenous neurogenesis appeared to be confined to a reduction in the generation of new neurons in the dentate gyrus of patients with AD and severe neurofibrillary tangle pathology and were accompanied by changes in the glia load. These data suggest that intervention enhancing endogenous neurogenesis may be a potential therapeutic target in AD.
Objective To develop a simple and efficient method for producing homogeneous populations of monocytes and macrophages from human embryonic stem cells (hES). Materials and Methods Human embryonic stem ...cell lines KCL001, KCL002, and HUES-2 were differentiated into monocytes by coculture-free differentiation with two growth factors using a three-step method. The method involved embryoid body (EB) formation in hES media, directed differentiation with macrophage colony-stimulating factor and interleukin (IL)-3, and harvest of nonadherent monocytes from the culture supernatants. hES monocytes (esMCs) were analyzed by microscopy, flow cytometry, transcriptome analysis, and tested for the ability to differentiate into macrophages. hES monocyte–derived macrophages (esMDM) were analyzed for phagocytosis and endocytosis by microscopy and flow cytometry, cytokine secretion by multiplex cytokine assay, and for interferon (IFN)-γ and IL-4 activation by flow cytometry. Results Homogeneous esMCs (>90% CD14-positive) that did not require any additional purification steps were produced after 18.7 ± 7.7 days (mean ± SD, n = 19). Production continued for several months when growth factors were replaced, with a total yield of 3.4 × 105 ± 2.0 esMCs (mean ± SD, n = 9) per EB. Transcriptome analysis of the esMC and the esMDM revealed a distinct myeloid signature that correlated with primary adult blood–derived monocytes and spleen tissue samples but not with other tissue samples tested. We found that esMCs and esMDMs expressed well-defined markers of the mononuclear phagocyte system including PU-1, C/EBPα, EMR1, and EMR2, MPEG1, CD1c, CD4, CD18, CD32, CD33, CD68, cathepsins and serine carboxypeptidase. Finally, esMCs differentiated into functional macrophages that could endocytose acetylated low-density lipoprotein, phagocytose opsonized yeast particles, secrete specific cytokines in response to lipopolysaccharide, and be activated differentially with IFN-γ and IL-4. Conclusions We have developed a simple and efficient method for producing homogeneous populations of monocytes and macrophages from hES cells. esMCs have a myeloid signature and can differentiate into functional macrophages. The method should prove useful in answering experimental questions regarding monocyte and macrophage development and biology.
Altered neurogenesis in Alzheimer's disease Ziabreva, Iryna; Perry, Elaine; Perry, Robert ...
Journal of psychosomatic research,
09/2006, Letnik:
61, Številka:
3
Journal Article
Recenzirano
Exciting preliminary work indicates an increase in progenitor activity in the subgranular zone of the dentate gyrus of people with Alzheimer's disease (AD) compared to that of controls. We examine ...progenitor activity in the other main progenitor niche, the subventricular zone (SVZ), as well as potential associations with key pathological and neurochemical substrates.
Immunocytochemistry techniques utilizing nestin and Musashi1 antibodies were used to examine progenitor activity in the SVZ and to enable comparisons between seven patients with AD and seven controls, based upon the quantification of the percentage area covered, using the Image Pro Plus v.4.1 image analysis system. AD pathology was staged using the Consortium to Establish a Registry for Alzheimer's Disease and Braak criteria. Choline acetyl transferase (ChAT) was measured in the temporal cortex as an indication of the severity of cortical cholinergic deficits. Glial fibrillary acidic protein (GFAP) was used to label astrocytes.
There was a significant ninefold decrease (
Z=2.2,
P=.046) of Musashi1 immunoreactivity in the SVZ of patients with AD in comparison with that of controls, but there was a significant increase in nestin immunoreactivity in the same region (
Z=2.2,
P=.028) without any significant change in GFAP immunoreactivity. Reduced ChAT enzymatic activity was the main association of Musashi immunoreactivity (
R=−.90,
P=.03).
The current results indicate a significant reduction of progenitor cells (as labeled by Musashi1) in the SVZ of patients with AD, but an increase in GFAP-negative astrocyte-like cells with progenitor characteristics. Cortical cholinergic loss was strongly associated with the reduction of progenitors, with potential implications of important treatment targets.
Since human embryonic stem cells (hESCs) were first isolated and cultured nearly 15 years ago, stem cell biology has been a promising and fast-moving area of research. Improved clinical predictivity ...in drug development, use in assays to personalise medicine effectively and as the foundation for cell-based therapies are all areas where stem cells can play an important role. But with opportunities come challenges and it is vital that the field of stem cells continues to progress to achieve its potential. This article outlines the measures the Cell Technologies group at GE Healthcare Life Sciences are taking, along with its collaborators in academia, industry and the clinic, to advance stem cell tools and technologies, as well as identifying some future challenges for stem cell research, drug discovery, cell therapy and regenerative medicine.
Increased endogenous neurogenesis has a significant regenerative role in many experimental models of cerebrovascular diseases, but there have been very few studies in humans. We therefore examined ...whether there was evidence of altered endogenous neurogenesis in an 84-year-old patient who suffered a cerebrovascular accident 1 week prior to death. Using antibodies that specifically label neural stem/neural progenitor cells, we examined the presence of immunopositive cells around and distant from the infarcted area, and compared this with a control, age-matched individual. Interestingly, a large number of neural stem cells, vascular endothelial growth factor-immunopositive cells and new blood vessels were observed only around the region of infarction, and none in the corresponding brain areas of the healthy control. In addition, an increased number of neural stem cells was observed in the neurogenic region of the lateral ventricle wall. Our results suggest increased endogenous neurogenesis associated with neovascularization and migration of newly-formed cells towards a region of cerebrovascular damage in the adult human brain and highlight possible mechanisms underlying this process.
The COVID-19 pandemic has significantly impacted the way of life worldwide and continues to bring high mortality rates to at-risk groups. Patients who develop severe COVID-19 pneumonia, often ...complicated with ARDS, are left with limited treatment options with no targeted therapy currently available. One of the features of COVID-19 is an overaggressive immune reaction that leads to multiorgan failure. Mesenchymal stromal cell (MSC) treatment has been in development for various clinical indications for over a decade, with a safe side effect profile and promising results in preclinical and clinical trials. Therefore, the use of MSCs in COVID-19-induced respiratory failure and ARDS was a logical step in order to find a potential treatment option for the most severe patients. In this review, the main characteristics of MSCs, their proposed mechanism of action in COVID-19 treatment and the effect of this therapy in published case reports and clinical trials are discussed.
Abstract Since groundbreaking studies demonstrated the presence of progenitor cells in the adult human brain, there have been intense interests in their potential therapeutic application, but to date ...only limited data has been obtained in man. An immunohistological study was performed in order to examine neurogenesis in both the subventricular and peri-infarct zones of vascular dementia patients compared to age-matched controls. The results were striking, showing a significant increase of progenitor cells in both the subventricular zone and in peri-infarct area in patients with vascular dementia compared to controls, which was sustained even in patients with infarcts occurring more than three months prior to autopsy. Moreover, the peri-infarct response appeared to be unified with that of the subventricular zone via a stream of cells, with some of them differentiating into immature neurons. We conclude that neurogenesis is stimulated in vascular dementia patients and, specifically, in patients with visible infarcts. Progenitors may migrate from the neurogenic niche to areas of infarction and differentiate into neurons, even three months after cerebrovascular damage, thus implicating the feasibility of enhancing neurogenesis as a novel treatment approach.
Human embryonic stem (hES) cells and fetal mesenchymal stem cells (fMSC) offer great potential for regenerative therapy strategies. It is therefore important to characterise the properties of these ...cells in vitro. One major way the environment impacts on cellular physiology is through changes to epigenetic mechanisms. Genes subject to epigenetic regulation via genomic imprinting have been characterised extensively. The integrity of imprinted gene expression therefore provides a measurable index for epigenetic stability. Allelic expression of 26 imprinted genes and DNA methylation at associated differentially methylated regions (DMRs) was measured in fMSC and hES cell lines. Both cell types exhibited monoallelic expression of 13 imprinted genes, biallelic expression of six imprinted genes, and there were seven genes that differed in allelic expression between cell lines. fMSCs exhibited the differential DNA methylation patterns associated with imprinted expression. This was unexpected given that gene expression of several imprinted genes was biallelic. However, in hES cells, differential methylation was perturbed. These atypical methylation patterns did not correlate with allelic expression. Our results suggest that regardless of stem cell origin, in vitro culture affects the integrity of imprinted gene expression in human cells. We identify biallelic and variably expressed genes that may inform on overall epigenetic stability. As differential methylation did not correlate with imprinted expression changes we propose that other epigenetic effectors are adversely influenced by the in vitro environment. Since DMR integrity was maintained in fMSC but not hES cells, we postulate that specific hES cell derivation and culturing practices result in changes in methylation at DMRs.
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