- The CXCR4 antagonist AMD3100 promotes mesenchymal stem cell mobilization in rats preconditioned with the hypoxia-mimicking agent cobalt chloride. [Journal Article]
- SCStem Cells Dev 2018 Feb 13
- Mobilization of mesenchymal stem cells (MSCs) is an attractive strategy for cell therapy. Our previous study demonstrated that MSCs can be mobilized in circulating blood by short-term hypoxia and hyp...
Mobilization of mesenchymal stem cells (MSCs) is an attractive strategy for cell therapy. Our previous study demonstrated that MSCs can be mobilized in circulating blood by short-term hypoxia and hypoxia-inducible factor-1α (HIF-1α) is essential for MSC mobilization. In the present study, the effect of the hypoxia mimicking agent CoCl2 was examined on MSC mobilization. The results indicated that the frequency of circulating MSCs increased slightly by administration of CoCl2. However, the mobilization efficiency was low. Considering the critical role of stromal cell-derived factor-1α (SDF-1)/CXCR4 axis in the regulation of MSC migration, the effects of granulocyte colony-stimulating factor (G-CSF) and the CXCR4 antagonist AMD3100 were investigated on MSC mobilization. The experiments were notably demonstrated in animals preconditioned with CoCl2. The frequency of colony-forming unit fibroblast (CFU-Fs) and the proportion of CD45-CD90+ cells did not significantly increase in the peripheral blood of rats treated with G-CSF and/or AMD3100 alone. The concomitant administration of G-CSF with CoCl2 could not stimulate the release of MSCs. However, AMD3100 dramatically increased MSC mobilization efficiency in rats pretreated with CoCl2. Furthermore, we identified and compared the multi-lineage differentiation capacities of MSCs derived from BM (BM-MSCs) and mobilized PB (PB-MSCs). The results indicated that PB-MSCs exhibited higher osteogenic potential and lower adipogenic differentiation compared with BM-MSCs. The findings may inform studies investigating mechanisms of the regulation of MSC mobilization and can aid the development of clinically useful therapeutic agents.
- Angiogenic activity of human adipose-derived mesenchymal stem cells under simulated microgravity. [Journal Article]
- SCStem Cells Dev 2018 Feb 12
- Multipotent mesenchymal stem/stromal cells (MSCs) are strongly involved in tissue homeostasis mainly through paracrine regulation. Here we examined the influence of simulated microgravity on the angi...
Multipotent mesenchymal stem/stromal cells (MSCs) are strongly involved in tissue homeostasis mainly through paracrine regulation. Here we examined the influence of simulated microgravity on the angiogenic potential of adipose-derived MSCs (ASCs). The conditioned medium (CM) from RPM-exposed ASCs stimulated the formation of vessel network in ovo, endothelial cell (EC) capillary-like network ex ovo and non-directed EC migration in vitro. These effects were driven by alteration of both angiogenesis-related gene and protein expression. The elevation of angiogenic regulators Serpin E1, Serpin F1, IGFBP, VEGF, IL-8 was detected in ASC-CM after 3D-clinorotation. Besides, transcription of genes encoded growth factors with proangiogenic activity were upregulated including VEGF-с and VEGF-a. These data evidenced that besides direct effect on ECs, microgravity could provoke MSC-mediating specific microenvironment for ECs supporting their functions i.e. proliferation and migration via increased production of IL-8 and VEGF as well as other paracrine factors involved in angiogenesis regulation.
- Cell density-dependent cytological stage profile and its application for a screen of cytostatic agents active towards Leukemic Stem Cells. [Journal Article]
- SCStem Cells Dev 2018 Feb 12
- Proliferation and expansion of leukemia is driven by Leukemic Stem Cells (LSCs). Multidrug resistance (MDR) of LSCs is one of the main reasons of failure and relapses in Acute Myeloid Leukemia (AML) ...
Proliferation and expansion of leukemia is driven by Leukemic Stem Cells (LSCs). Multidrug resistance (MDR) of LSCs is one of the main reasons of failure and relapses in Acute Myeloid Leukemia (AML) treatment. Here we show that maintaining HL-60 at low cell culture density or applying a 240-day treatment with anthrapyridazone (BS-121) increased the percentage of primitive cells which include LSCs determining the overall stage profile. This change manifested in: morphology, expression of both cell surface markers and redox-state proteins as well as mitochondrial potential. Moreover, four sublines were generated, each with unique and characteristic stage profile and cytostatic sensitivity. Cell density-induced culture alterations (affecting stage profiles) were exploited in a screen of anthrapyridazones. Amongst the compound tested, C-123 was the most potent against primitive cell stages while generating relatively low amounts of ROS. Furthermore, it had low toxicity<i> in vivo</i> and weakly affected blood morphology of healthy mice. The cell density-dependent stage profiles could be utilized in preliminary drug screens for activity against LSCs or in construction of patient-specific platforms to find drugs effective in case of AML relapse (drug extrapolation). The correlation between ROS generation in differentiated cells and toxic effect observed in HL-60 has a potential application in myelotoxicity predictions. The discovered properties of C 123 indicate its potential application in AML treatment, specifically in conditioned myeloablation preceding allogenic transplantation and/or <i>ex vivo</i> treatment preceding autologous transplantation.
- Exosomes derived from hypoxia-treated human adipose mesenchymal stem cells enhance angiogenesis through the PKA signaling pathway. [Journal Article]
- SCStem Cells Dev 2018 Feb 07
- Angiogenesis is a complicated and sequential process that plays an important role in different physiological processes. Mesenchymal stem cells (MSCs), which are pluripotent stem cells, are widely use...
Angiogenesis is a complicated and sequential process that plays an important role in different physiological processes. Mesenchymal stem cells (MSCs), which are pluripotent stem cells, are widely used for the treatment of ischemic and traumatic diseases, and exosomes derived from these cells can also promote angiogenesis. Therefore, we aimed to uncover mechanisms to improve MSC exosome-mediated angiogenesis. For this study, we isolated human adipose-derived mesenchymal stem cells (hAD-MSCs) and assessed differentiation ability and markers. Cells were divided into hypoxia- (H-MSCs) and normoxia-treated (N-MSC) mesenchymal stem cells, and exosomes were extracted by ultrafiltration. Exosomes (100 µg /ml) from H-MSCs and N-MSCs were added to human umbilical vein endothelial cells (HUVECs). Exosome uptake and the ability of endothelial cells to form tubes were detected in real time. Protein samples were collected at different time points to detect the expression of inhibitors (Vash1) and enhancers (Angpt1 and Flk1) of angiogenesis; we also assessed their related signaling pathways. We found that exosomes from the hypoxia group were more easily taken up by HUVECs; further, their angiogenesis stimulatory activity was also significantly enhanced compared to that with exosomes from the normoxia group. HUVECs exposed to exosomes from H-MSCs significantly upregulated angiogenesis-stimulating genes and deregulated angiogenesis-inhibitory genes. The expression of vascular endothelial growth factor (VEGF) and activation of the PKA signaling pathway in HUVECs were significantly increased by hypoxia-exposed exosomes. Moreover, a PKA inhibitor was shown to significantly suppress angiogenesis. Finally, we concluded that hypoxia-exposed exosomes derived from hAD-MSCs can improve angiogenesis by activating the PKA signaling pathway and promoting the expression of VEGF. These results could be used to uncover safe and effective treatments for traumatic diseases.
- Neuropilin-1 Mediated Arterial Differentiation of Murine Pluripotent Stem Cells. [Journal Article]
- SCStem Cells Dev 2018 Feb 07
- Pluripotent stem cell derived endothelial cells (ECs) have great potential to be used in vascular therapy or tissue engineering. It is also much desired to obtain arterial or venous ECs for specific ...
Pluripotent stem cell derived endothelial cells (ECs) have great potential to be used in vascular therapy or tissue engineering. It is also much desired to obtain arterial or venous ECs for specific applications. Factors that are critical for the proper arterial or venous differentiation from pluripotent stem cells are still need to be understood. Here, we aim to investigate this problem deeper by examining neuropilin-1 (Nrp1), an early arterial marker that may be critical for arterial cell fate commitment. Using murine embryonic stem cells as the model system, this study investigates the neuropilin-1 (Nrp1) expression during the differentiation of pluripotent stem cells toward a vascular progenitor population. We hypothesize that Nrp1, an early arterial marker present in a developing embryo, may be more responsive when further induced in vitro towards an arterial fate. We developed a two-step differentiation approach that yielded a large percentage of Nrp1+ vascular progenitor cells (VPCs) and investigated their potential to become arterial ECs. We have defined the culture parameters that contribute greatly to the emergence of Nrp1+ VPCs: certain soluble factors especially Wnt and BMP4, early cell-cell contact, and hypoxia. Subsequent isolation of this population demonstrated a highly proliferative and network-forming behavior. The Nrp1+ VPCs exhibited increased gene expression of several Notch pathway-related arterial markers compared to Nrp1- VPCs. Most importantly, Nrp1+ VPCs demonstrated dramatically greater response to hemodynamic stimuli by up-regulating many arterial markers while Nrp1- VPCs have very little response. Surprisingly, these differences between Nrp1+ and Nrp1- VPCs are not evident with VEGF treatment. Our data suggest that Nrp1+ VPCs may serve as the arterial progenitor by enhanced response to hemodynamic flow but not to VEGF, whereas Nrp1- VPCs lack the plasticity to become arterial ECs. The findings of this research indicate that Nrp1+ VPCs in the murine model act as an important step in the arterial differentiation process.
- Is Parkinson's disease a neurodevelopmental disorder and will brain organoids help us to understand it? [Journal Article]
- SCStem Cells Dev 2018 Feb 07
- Parkinson's disease is the second most common neurodegenerative disorder after Alzheimer's disease. The incidence of PD cases increases with age, accordingly classically PD is considered to be an age...
Parkinson's disease is the second most common neurodegenerative disorder after Alzheimer's disease. The incidence of PD cases increases with age, accordingly classically PD is considered to be an age associated neurodegenerative disease. In this review the hypothesis that PD is actually a neurodevelopmental disorder that is compensated for long time, will be discussed. However, patients who suffer from Parkinson's disease typically do not show symptoms early in their live. This implies that, if the hypothesis that PD has a significant neurodevelopmental component is correct, the developmental defects are compensated for long time. Furthermore, these developmental defects might not causally lead to the disease but increase the susceptibility for disease onset after a "second hit". In this logic deregulated developmental processes might represent the "first hit". Even a minor developmental defect could lead to a reduced compensatory capacity or reduced fault tolerance of the entire system. In such a case of an already imbalanced system one or more additional hits could perturb the entire system sufficiently to bring it out of balance and lead to the pathology and symptoms which we classify as PD. However, if the developmental hypothesis and the "multiple hit" hypothesis are correct an early diagnosis of these developmental defects might allow the start of a therapy for at risk individuals before disease pathology becomes severe and before symptoms occur. Modern stem cell technologies, including the generation of personalized brain organoids, might play an important role in these strategies.
- Human Umbilical Cord Perivascular Cells and Human Bone Marrow Mesenchymal Stromal Cells Transplanted Intramuscularly Respond to a Distant Source of Inflammation. [Journal Article]
- SCStem Cells Dev 2018 Feb 05
- Intravenously administered mesenchymal stromal cells (MSCs) are rapidly entrapped in the lungs, where they display an anti-inflammatory phenotype. Intramuscular (IM) delivery provides an increased MS...
Intravenously administered mesenchymal stromal cells (MSCs) are rapidly entrapped in the lungs, where they display an anti-inflammatory phenotype. Intramuscular (IM) delivery provides an increased MSC dwell-time, which could result in a sustained modulation of an inflammatory milieu. We studied the therapeutic effects of IM delivered MSCs to treat a distant (contralateral) inflammation, and compared the efficacy of neonatal (umbilical cord) and adult bone marrow (BM) MSCs. Inflammation decreased over 48h but neonatal cells showed an earlier response than BMMSCs. Tumor necrosis factor induced gene-6 (TSG-6), was released at the site of MSC delivery, while neutrophil infiltration was abrogated and inflammation reduced at the contralateral site. MSCs did not distribute to organs, nor to the site of inflammation. Thus, IM delivery presents a promising alternative for the treatment of inflammation, and neonatal MSCs may represent a stronger candidate than those derived from adult bone marrow to treat inflammatory diseases.
- Simplified footprint-free Cas9/CRISPR editing of cardiac-associated genes in human pluripotent stem cells. [Journal Article]
- SCStem Cells Dev 2018 Feb 05
- Modelling disease with hPSCs is hindered because the impact on cell phenotype from genetic variability between individuals can be greater than from the pathogenic mutation. While 'footprint-free' Cas...
Modelling disease with hPSCs is hindered because the impact on cell phenotype from genetic variability between individuals can be greater than from the pathogenic mutation. While 'footprint-free' Cas9/CRISPR editing solves this issue, existing approaches are inefficient or lengthy. Here, a simplified PiggyBac strategy shortened hPSC editing by 2 weeks and required one round of clonal expansion and genotyping rather than two, with similar efficiencies to the longer conventional process. Success was shown across 4 cardiac-associated loci (ADRB2, GRK5, RYR2, ACTC1) by genomic cleavage and editing efficiencies of 8-93% and 8-67%, respectively, including mono- and/or bi-allelic events. Pluripotency was retained, as was differentiation into high purity cardiomyocytes (CMs; 88-99%). Using the GRK5 isogenic lines as an exemplar, chronic stimulation with the b-adrenoceptor agonist, isoprenaline, reduced beat rate in hPSC-CMs expressing GRK5-Q41 but not GRK5-L41; this was reversed by the b-blocker, propranolol. This simplified, footprint-free approach will be useful for mechanistic studies.
- In Vitro Modeling of Leucine-Rich Repeat Kinase 2 (LRRK2) G2019S-Mediated Parkinson's Disease Pathology. [Journal Article]
- SCStem Cells Dev 2018 Feb 05
- Leucine-rich repeat kinase 2 (LRRK2) G2019S (glycine to serine) is the most common mutation associated with sporadic and familial Parkinson's disease (PD) with 80% penetrance by age 70. This mutation...
Leucine-rich repeat kinase 2 (LRRK2) G2019S (glycine to serine) is the most common mutation associated with sporadic and familial Parkinson's disease (PD) with 80% penetrance by age 70. This mutation is found worldwide, with up to 40% of individuals in the North African Arab population carrying the mutation. Induced pluripotent stem cells (iPSCs) derived from fibroblasts of patients carrying the LRRK2 G2019S mutation have been a critical source of cells for generating dopaminergic neurons and studying G2019S-related pathology. These studies have elucidated LRRK2-related mechanisms of mitochondrial dysregulation, increased reactive oxygen species, truncated and simplified neurites, and cell death. These phenotypes are thought to result from the G2019S mutation increasing substrate access and therefore increasing the catalytic rate of the serine/threonine kinase. In this article, we critically review the contributions of in vitro modeling to the current knowledge on LRRK2 G2019S. We also analyze the role of patient-derived cell lines for the identification and validation of therapeutic targets, emphasizing their importance as part of a 3R approach to translational research and personalized medicine.
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- Imprinting status of two human parthenogenetic embryonic stem cell lines: analysis of 63 imprinted genes expression levels in undifferentiated and early differentiated stages. [Journal Article]
- SCStem Cells Dev 2018 Feb 05
- Human parthenogenetic embryonic stem cells (hPESCs) represent a source of histocompatible tissues for transplantation and carry two copies of the maternally genome, but lack the paternally genome. In...
Human parthenogenetic embryonic stem cells (hPESCs) represent a source of histocompatible tissues for transplantation and carry two copies of the maternally genome, but lack the paternally genome. In this study, we selected 63 known human imprinted genes to investigate the imprinting status of hPESC. The expression level of these genes including 27 maternally and 36 paternally imprinted were illustrated in hPESC and human embryonic stem cells derived from fertilized embryos (hESC) cell lines. The expression activity changes of these genes were analyzed in undifferentiated and early differentiated hPESC cell lines. In addition, the methylation status of four differentially methylated regions (DMRs) of the imprinted genes was analyzed in undifferentiated and early differentiated hPESC and hESC cell lines. As results, we found that all the maternally imprinted genes were expressed at similar level in the undifferentiated hPESC cell lines and the hESC cell lines, except ZNF264 and ATP10A. 21 analyzed paternal imprinted genes were expressed at the same level in two separated hPESC cell lines as well as compared to the hESC lines, whereas 15 other paternal imprinted genes were significantly downregulated or inactivated in hPESC cell lines as compared to the hESC cell line. During prolonged passage, the expression levels of the majority of imprinted genes remained stable in two hPESC lines. The four DMRs including PEG3/ZIM2 (DMRs), SNURF/SNRPN DMRs and KVDMR1 DMRs are highly methylated in the genes of two undifferentiated hPESCs and its embryonic bodies (EBs), whereas in the genes of the undifferentiated hESCs and its EBs are half methylated. During the early differentiation stage, the imprinted genes showed the same expression trend and the expression levels of H19, IGF2, SLC22A2, SLC22A3/SLC22A18 and CPA4 were significantly up-regulated in both hPESC cell lines. As conclusion, hPESCs show a substantial degree of epigenetic stability with respect to some imprinted genes.