scholarly journals Generation of Inducible CRISPRi and CRISPRa Human Stromal/Stem Cell Lines for Controlled Target Gene Transcription during Lineage Differentiation

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Li Chen ◽  
Kaikai Shi ◽  
Weimin Qiu ◽  
Lars Aagaard ◽  
Moustapha Kassem
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Transfection Efficiency ◽  
Transcription Activator ◽  
Lineage Differentiation ◽  
Stem Cell Lines ◽  
Stromal Stem Cells ◽  
Activate Gene ◽  
Cell Growth Kinetics

Background. Human bone marrow stromal/stem cells (hMSCs, also known as the skeletal stem cells or mesenchymal stem cells) are being employed to study lineage fate determination to osteoblasts, adipocytes, and chondrocytes. However, mechanistic studies employing hMSC have been hampered by the difficulty of deriving genetically modified cell lines due to the low and unstable transfection efficiency. Methods. We infected hMSC with a CRISPR/Cas9 lentivirus system, with specific inducible dCas9-coupled transcription activator or repressor: dCas9-KRAB or dCas9-VP64, respectively, and established two hMSC lines (hMSC-CRISPRi and hMSC-CRISPRa) that can inhibit or activate gene expression, respectively. The two cell lines showed similar cell morphology, cell growth kinetics, and similar lineage differentiation potentials as the parental hMSC line. The expression of KRAB-dCas9 or VP64-dCas9 was controlled by the presence or absence of doxycycline (Dox) in the cell culturing medium. To demonstrate the functionality of the dCas9-effector hMSC system, we tested controlled expression of alkaline phosphatase (ALP) gene through transfection with the same single ALP sgRNA. Results. In the presence of Dox, the expression of ALP showed 60-90% inhibition in hMSC-CRISPRi while ALP showed more than 20-fold increased expression in hMSC-CRISPRa. As expected, the ALP was functionally active and the cells showed evidence for inhibition or enhancement of in vitro osteoblast differentiation, respectively. Conclusion. hMSC-CRISPRi and hMSC-CRISPRa are useful resources to study genes and genetic pathways regulating lineage-specific differentiation of hMSC.

scholarly journals Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells

2015 ◽  
Vol 370 (1680) ◽  
pp. 20140365 ◽  
Author(s):  
Maria Rostovskaya ◽  
Nicholas Bredenkamp ◽  
Austin Smith
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Human Pluripotent Stem Cells ◽  
Type I ◽  
Pancreatic Progenitors ◽  
Stem Cell Lines

Human pluripotent stem cells can in principle be used as a source of any differentiated cell type for disease modelling, drug screening, toxicology testing or cell replacement therapy. Type I diabetes is considered a major target for stem cell applications due to the shortage of primary human beta cells. Several protocols have been reported for generating pancreatic progenitors by in vitro differentiation of human pluripotent stem cells. Here we first assessed one of these protocols on a panel of pluripotent stem cell lines for capacity to engender glucose sensitive insulin-producing cells after engraftment in immunocompromised mice. We observed variable outcomes with only one cell line showing a low level of glucose response. We, therefore, undertook a systematic comparison of different methods for inducing definitive endoderm and subsequently pancreatic differentiation. Of several protocols tested, we identified a combined approach that robustly generated pancreatic progenitors in vitro from both embryo-derived and induced pluripotent stem cells. These findings suggest that, although there are intrinsic differences in lineage specification propensity between pluripotent stem cell lines, optimal differentiation procedures may consistently direct a substantial fraction of cells into pancreatic specification.

Epigenetic regulation in stem cell development, cell fate conversion, and reprogramming

2015 ◽  
Vol 6 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Kazuyuki Ohbo ◽  
Shin-ichi Tomizawa
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Transcription Factors ◽  
Cell Fate ◽  
Cell Lines ◽  
Cell Fate Decisions ◽  
Micro Rnas ◽  
Stem Cell Lines ◽  
Cell Fate Conversion

AbstractStem cells are identified classically by an in vivo transplantation assay plus additional characterization, such as marker analysis, linage-tracing and in vitro/ex vivo differentiation assays. Stem cell lines have been derived, in vitro, from adult tissues, the inner cell mass (ICM), epiblast, and male germ stem cells, providing intriguing insight into stem cell biology, plasticity, heterogeneity, metastable state, and the pivotal point at which stem cells irreversibly differentiate to non-stem cells. During the past decade, strategies for manipulating cell fate have revolutionized our understanding about the basic concept of cell differentiation: stem cell lines can be established by introducing transcription factors, as with the case for iPSCs, revealing some of the molecular interplay of key factors during the course of phenotypic changes. In addition to de-differentiation approaches for establishing stem cells, another method has been developed whereby induced expression of certain transcription factors and/or micro RNAs artificially converts differentiated cells from one committed lineage to another; notably, these cells need not transit through a stem/progenitor state. The molecular cues guiding such cell fate conversion and reprogramming remain largely unknown. As differentiation and de-differentiation are directly linked to epigenetic changes, we overview cell fate decisions, and associated gene and epigenetic regulations.

scholarly journals Loss of proliferative capacity in immunohemopoietic stem cells caused by serial transplantation rather than aging.

1978 ◽  
Vol 147 (5) ◽  
pp. 1526-1531 ◽  
Author(s):  
D E Harrison ◽  
C M Astle ◽  
J A Delaittre
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Lines ◽  
Cell Types ◽  
Colony Growth ◽  
Normal Function ◽  
Stem Cell Lines ◽  
Marrow Stem Cell ◽  
Serial Transplantation

Marrow stem cell lines from old donors and those from young controls gave equally rapid rates of colony growth on spleens of irradiated mice. Old and young stem cell lines competed equally well with chromosomally marked marrow stem cells from a young donor in producing cell types that are stimulated by bleeding; old cells competed 70% as well as young in producing cell types stimulated by phytohemagglutinin (PHA) in vitro. After a single serial transplantation, the rates of colony growth declined 1.5- to 2.5-fold, and the ability to compete declined 2- to 4-fold for bleeding-stimulated and 4- to 10-fold for PHA-stimulated cells. Thus, immediate stem cell proliferative capacities decline much more after one serial transplantation than after a lifetime of normal function.

scholarly journals Patentability of the Human Embryonic Stem Cell Lines: A Legal and Ethical Aspect

2020 ◽  
Author(s):  
Tansu Sayar Kanyış ◽  
Ezgi Arslan ◽  
Oğuzhan Kanyış
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Cell Lines ◽  
Human Embryo ◽  
Human Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Point Of View ◽  
Stem Cell Lines

In this study, patentability of the human embryonic stem cell lines has discussed in the legal and ethical perspectives. In vitro human embryonic stem cells can be defined as body parts that are departed from the body. Human embryonic stem cell lines are constituted of differentiated self-renewal pluripotent stem cells, which means they have no characteristics to become a human-being. However, interpreting the terms like human embryo and right to property widely can cause the human embryonic stem cell lines are misunderstood as unpatentable. For our point of view, giving the human embryo the protections of both personal rights of the donor and the right to property of the owner of the invention does not reduce the legal/moral status of the human embryo. Besides, the obligations which these rights imposes to their owners, such as the principle of human dignity and prohibition of financial gain can protect the human embryo in a better way.

scholarly journals Generation and characterization of stable pig pregastrulation epiblast stem cell lines

Cell Research ◽  
2021 ◽  
Author(s):  
Minglei Zhi ◽  
Jinying Zhang ◽  
Qianzi Tang ◽  
Dawei Yu ◽  
Shuai Gao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Large Scale ◽  
Biological Research ◽  
Marker Genes ◽  
Stem Cell Lines ◽  
Cloned Gene

AbstractPig epiblast-derived pluripotent stem cells are considered to have great potential and broad prospects for human therapeutic model development and livestock breeding. Despite ongoing attempts since the 1990s, no stably defined pig epiblast-derived stem cell line has been established. Here, guided by insights from a large-scale single-cell transcriptome analysis of pig embryos from embryonic day (E) 0 to E14, specifically, the tracing of pluripotency changes during epiblast development, we developed an in vitro culture medium for establishing and maintaining stable pluripotent stem cell lines from pig E10 pregastrulation epiblasts (pgEpiSCs). Enabled by chemical inhibition of WNT-related signaling in combination with growth factors in the FGF/ERK, JAK/STAT3, and Activin/Nodal pathways, pgEpiSCs maintain their pluripotency transcriptome features, similar to those of E10 epiblast cells, and normal karyotypes after more than 240 passages and have the potential to differentiate into three germ layers. Strikingly, ultradeep in situ Hi-C analysis revealed functional impacts of chromatin 3D-spatial associations on the transcriptional regulation of pluripotency marker genes in pgEpiSCs. In practice, we confirmed that pgEpiSCs readily tolerate at least three rounds of successive gene editing and generated cloned gene-edited live piglets. Our findings deliver on the long-anticipated promise of pig pluripotent stem cells and open new avenues for biological research, animal husbandry, and regenerative biomedicine.

scholarly journals Aspirin enhances the therapeutic efficacy of cisplatin in oesophageal squamous cell carcinoma by inhibition of putative cancer stem cells

2021 ◽  
Author(s):  
Zhigeng Zou ◽  
Wei Zheng ◽  
Hongjun Fan ◽  
Guodong Deng ◽  
Shih-Hsin Lu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Squamous Cell Carcinoma ◽  
Cancer Stem Cells ◽  
Cell Carcinoma ◽  
Cell Lines ◽  
Squamous Cell ◽  
High Throughput Sequencing ◽  
Combined Treatment ◽  
Oesophageal Squamous Cell Carcinoma

Abstract Background Cancer stem cells (CSCs) are related to the patient’s prognosis, recurrence and therapy resistance in oesophageal squamous cell carcinoma (ESCC). Although increasing evidence suggests that aspirin (acetylsalicylic acid, ASA) could lower the incidence and improve the prognosis of ESCC, the mechanism(s) remains to be fully understood. Methods We investigated the role of ASA in chemotherapy/chemoprevention in human ESCC cell lines and an N-nitrosomethylbenzylamine-induced rat ESCC carcinogenesis model. The effects of combined treatment with ASA/cisplatin on ESCC cell lines were examined in vitro and in vivo. Sphere-forming cells enriched with putative CSCs (pCSCs) were used to investigate the effect of ASA in CSCs. Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) was performed to determine the alterations in chromatin accessibility caused by ASA in ESCC cells. Results ASA inhibits the CSC properties and enhances cisplatin treatment in human ESCC cells. ATAC-seq indicates that ASA treatment results in remarkable epigenetic alterations on chromatin in ESCC cells, especially their pCSCs, through the modification of histone acetylation levels. The epigenetic changes activate Bim expression and promote cell death in CSCs of ESCC. Furthermore, ASA prevents the carcinogenesis of NMBzA-induced ESCC in the rat model. Conclusions ASA could be a potential chemotherapeutic adjuvant and chemopreventive drug for ESCC treatment.

scholarly journals Adipose-derived stromal/stem cells improve epidermal homeostasis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Mariko Moriyama ◽  
Shunya Sahara ◽  
Kaori Zaiki ◽  
Ayumi Ueno ◽  
Koichi Nakaoji ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wound Healing ◽  
Culture System ◽  
Wound Repair ◽  
Cell Types ◽  
Skin Wound ◽  
Epidermal Keratinocytes ◽  
Stromal Stem Cells ◽  
Collagen Vitrigel

AbstractWound healing is regulated by complex interactions between the keratinocytes and other cell types including fibroblasts. Recently, adipose-derived mesenchymal stromal/stem cells (ASCs) have been reported to influence wound healing positively via paracrine involvement. However, their roles in keratinocytes are still obscure. Therefore, investigation of the precise effects of ASCs on keratinocytes in an in vitro culture system is required. Our recent data indicate that the epidermal equivalents became thicker on a collagen vitrigel membrane co-cultured with human ASCs (hASCs). Co-culturing the human primary epidermal keratinocytes (HPEK) with hASCs on a collagen vitrigel membrane enhanced their abilities for cell proliferation and adhesion to the membrane but suppressed their differentiation suggesting that hASCs could maintain the undifferentiated status of HPEK. Contrarily, the effects of co-culture using polyethylene terephthalate or polycarbonate membranes for HPEK were completely opposite. These differences may depend on the protein permeability and/or structure of the membrane. Taken together, our data demonstrate that hASCs could be used as a substitute for fibroblasts in skin wound repair, aesthetic medicine, or tissue engineering. It is also important to note that a co-culture system using the collagen vitrigel membrane allows better understanding of the interactions between the keratinocytes and ASCs.

scholarly journals In Vitro Blood-Brain-Barrier Permeability and Cytotoxicity of Atorvastatin-Loaded Nanoformulation Against Glioblastoma in 2D and 3D Models

2019 ◽  
Author(s):  
Michael M Lübtow ◽  
Sabrina Oertner ◽  
Sabina Quader ◽  
Elisabeth Jeanclos ◽  
Alevtina Cubukova ◽  
...  
Keyword(s):  
Stem Cells ◽  
Oral Administration ◽  
Blood Brain Barrier ◽  
Cell Lines ◽  
Drug Loading ◽  
3D Models ◽  
Physicochemical Characteristics ◽  
Brain Barrier ◽  
Blood Brain

Inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase of the family of statins have been suggested as therapeutic options in various tumors. Atorvastatin is a statin with potential to cross the blood-brain-barrier, however, the concentrations necessary for a cytotoxic effect against cancer cells exceeds the concentration achievable via oral administration, which made the development of a novel atorvastatin formulation necessary. We characterized the drug loading and basic physicochemical characteristics of micellar atorvastatin formulations and tested their cytotoxicity against a panel of different glioblastoma cell lines. In addition, activity against tumor spheroids formed from mouse glioma and mouse cancer stem cells, respectively, was evaluated. Our results show good activity of atorvastatin against all tested cell lines. Interestingly, in the 3D models, growth inhibition was more pronounced for the micellar formulation compared to free atorvastatin. Finally, atorvastatin penetration across a blood-brain-barrier model obtained from human induced-pluripotent stem cells was evaluated. Our results suggest that the presented micelles may enable much higher serum concentrations than possible by oral administration, however, if transport across the blood-brain-barrier is sufficient to reach therapeutic atorvastatin concentration for the treatment of glioblastoma via intravenous administration remains unclear.<br>

scholarly journals Soft Substrate Maintains Proliferative and Adipogenic Differentiation Potential of human Mesenchymal Stem Cells on Long Term Expansion by Delaying Senescence

2018 ◽  
Author(s):  
Sanjay K. Kureel ◽  
Pankaj Mogha ◽  
Akshada Khadpekar ◽  
Vardhman Kumar ◽  
Rohit Joshi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Culture System ◽  
Human Mesenchymal Stem Cells ◽  
Population Doubling ◽  
Differentiation Potential ◽  
Lineage Differentiation ◽  
Poly Acrylamide

AbstractHuman mesenchymal stem cells (hMSCs), when cultured on tissue culture plate (TCP) for in vitro expansion, they spontaneously lose their proliferative capacity and multi-lineage differentiation potential. They also lose their distinct spindle morphology and become large and flat. After a certain number of population doubling, they enter into permanent cell cycle arrest, called senescence. This is a major roadblock for clinical use of hMSCs which demands large number of cells. A cell culture system is needed which can maintain the stemness of hMSCs over long term passages yet simple to use. In this study, we explore the role of substrate rigidity in maintaining stemness. hMSCs were serially passaged on TCP and 5 kPa poly-acrylamide gel for 20 population doubling. It was found that while on TCP, cell growth reached a plateau at cumulative population doubling (CPD) = 12.5, on 5 kPa gel, they continue to proliferate linearly till we monitored (CPD = 20). We also found that while on TCP, late passage MSCs lost their adipogenic potential, the same was maintained on soft gel. Cell surface markers related to MSCs were also unaltered. We demonstrated that this maintenance of stemness was correlated with delay in onset of senescence, which was confirmed by β-gal assay and by differential expression of vimentin, Lamin A and Lamin B. As preparation of poly-acrylamide gel is a simple, well established, and well standardized protocol, we believe that this system of cell expansion will be useful in therapeutic and research applications of hMSCs.One Sentence SummaryhMSCs retain their stemness when expanded in vitro on soft polyacrylamide gel coated with collagen by delaying senescence.Significance StatementFor clinical applications, mesenchymal stem cells (MSCs) are required in large numbers. As MSCs are available only in scarcity in vivo, to fulfill the need, extensive in vitro expansion is unavoidable. However, on expansion, they lose their replicative and multi-lineage differentiation potential and become senescent. A culture system that can maintain MSC stemness on long-term expansion, without compromising the stemness, is need of the hour. In this paper, we identified polyacrylamide (PAA) hydrogel of optimum stiffness that can be used to maintain stemness of MSCs during in vitro long term culture. Large quantity of MSCs thus grown can be used in regenerative medicine, cell therapy, and in treatment of inflammatory diseases.

Sign in / Sign up

Export Citation Format

Share Document