| 11. |
- Chen, Liangliang, et al.
(författare)
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User-Friendly Genetic Conditional Knockout Strategies by CRISPR/Cas9
- 2018
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Ingår i: STEM CELLS INTERNATIONAL. - : HINDAWI LTD. - 1687-966X .- 1687-9678.
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Tidskriftsartikel (refereegranskat)abstract
- Loss-of-function studies are critically important in gene functional analysis of model organisms and cells. However, conditional gene inactivation in diploid cells is difficult to achieve, as it involves laborious vector construction, multifold electroporation, and complicated genotyping. Here, a strategy is presented for generating biallelic conditional gene and DNA regulatory region knockouts in mouse embryonic stem cells by codelivery of CRISPR-Cas9 and short-homology-arm targeting vectors sequentially or simultaneously. Collectively, a simple and rapid method was presented to knock out any DNA element conditionally. This approach will facilitate the functional studies of essential genes and regulatory regions during development.
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| 12. |
- Dottori, Mirella, et al.
(författare)
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Stem Cells as In Vitro Models of Disease
- 2012
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Ingår i: Stem Cells International. - : Hindawi Limited. - 1687-966X .- 1687-9678. ; 2012
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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| 13. |
- Ekblad, A, et al.
(författare)
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Soft Tissue Repair with Easy-Accessible Autologous Newborn Placenta or Umbilical Cord Blood in Severe Malformations: A Primary Evaluation
- 2017
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Ingår i: Stem cells international. - : Hindawi Limited. - 1687-966X .- 1687-9678. ; 2017, s. 1626741-
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Tidskriftsartikel (refereegranskat)abstract
- Disrupted organogenesis leads to permanent malformations that may require surgical correction. Autologous tissue grafts may be needed in severe lack of orthotopic tissue but include donor site morbidity. The placenta is commonly discarded after birth and has a therapeutic potential. The aim of this study was to determine if the amnion from placenta or plasma rich of growth factors (PRGF) with mononuclear cells (MNC) from umbilical cord blood (UCB), collected noninvasively, could be used as bio-constructs for autologous transplantation as an easy-accessible no cell culture-required method. Human amnion and PRGF gel were isolated and kept in culture for up to 21 days with or without small intestine submucosa (SIS). The cells in the constructs showed a robust phenotype without induced increased proliferation (Ki67) or apoptosis (caspase 3), but the constructs showed decreased integrity of the amnion-epithelial layer at the end of culture. Amnion-residing cells in the SIS constructs expressed CD73 or pan-cytokeratin, and cells in the PRGF-SIS constructs expressed CD45 and CD34. This study shows that amnion and UCB are potential sources for production of autologous grafts in the correction of congenital soft tissue defects. The constructs can be made promptly after birth with minimal handling or cell expansion needed.
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| 14. |
- Erkers, T, et al.
(författare)
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Lymphocytes in Placental Tissues: Immune Regulation and Translational Possibilities for Immunotherapy
- 2017
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Ingår i: Stem cells international. - : Hindawi Limited. - 1687-966X .- 1687-9678. ; 2017, s. 5738371-
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Tidskriftsartikel (refereegranskat)abstract
- Immune modulation at the fetomaternal interface is crucial to ensure that the fetal allograft is not rejected. In the present review, the focus is to describe basic functions of lymphocyte populations and how they may contribute to fetomaternal immune regulation, as well as determining what proportions and effector functions of these cells are reported to be present in placental tissues in humans. Also explored is the possibility that unique cell populations at the fetomaternal interface may be targets for adoptive cell therapy. Increasing the understanding of immune modulation during pregnancy can give valuable insight into other established fields such as allogeneic hematopoietic stem cell transplantation and solid organ transplantation. In these settings, lymphocytes are key components that contribute to inflammation and rejection of either patient or donor tissues following transplantation. In contrast, an allogeneic fetus eludes rejection by the maternal immune system.
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| 15. |
- Gaballa, Ahmed, et al.
(författare)
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CD8(+)gamma delta T Cells Are More Frequent in CMV Seropositive Bone Marrow Grafts and Display Phenotype of an Adaptive Immune Response
- 2019
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Ingår i: STEM CELLS INTERNATIONAL. - : HINDAWI LTD. - 1687-966X .- 1687-9678. ; 2019
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Tidskriftsartikel (refereegranskat)abstract
- The role of gamma delta (gamma delta) T cells in human cytomegalovirus (HCMV) immune surveillance has been the focus of research interest for years. Recent reports have shown a substantial clonal proliferation of gamma delta T cells in response to HCMV, shedding light on the adaptive immune response of gamma delta T cells. Nevertheless, most efforts have focused on V delta 2(neg)gamma delta T cell subset while less attention has been given to investigate other less common gamma delta T cell subsets. In this regard, a distinct subpopulation of gamma delta T cells that expresses the CD8 coreceptor (CD8(+)gamma delta T cells) has not been thoroughly explored. Whether it is implicated in HCMV response and its ability to generate adaptive response has not been thoroughly investigated. In this study, we combined flow cytometry and immune sequencing of the TCR gamma-chain (TRG) to analyze in-depth bone marrow (BM) graft gamma delta T cells from CMV seropositive (CMV+) and CMV seronegative (CMV-) donors. We showed that the frequency of CD8(+)gamma delta T cells was significantly higher in CMV+ grafts compared to CMV- grafts (P<0.001). Further characterization revealed that CD8(+)gamma delta T cells from CMV+ grafts express V gamma 9(-) and preferentially differentiated from a naive to terminal effector memory phenotype (CD27(low/-)CD45RO(-)). In line with these findings, TRG immune sequencing revealed clonal focusing and reduced usage of the V gamma 9/JP gene segment in a CMV+ graft. Furthermore, CD8(+)gamma delta T cells showed an enhanced response to TCR/CD3 and cytokine stimulation in contrast to CD8(-)gamma delta T cells. We conclude that gamma delta T cells in BM grafts are reshaped by donor CMV serostatus and highlight the potential adaptive role of CD8(+)gamma delta T cells in HCMV immune response.
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| 16. |
- Ghosheh, Nidal, et al.
(författare)
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Highly Synchronized Expression of Lineage-Specific Genes during In Vitro Hepatic Differentiation of Human Pluripotent Stem Cell Lines
- 2016
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Ingår i: Stem Cells International. - : Hindawi Limited. - 1687-966X .- 1687-9678.
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Tidskriftsartikel (refereegranskat)abstract
- Human pluripotent stem cells- (hPSCs-) derived hepatocytes have the potential to replace many hepatic models in drug discovery and provide a cell source for regenerative medicine applications. However, the generation of fully functional hPSC-derived hepatocytes is still a challenge. Towards gaining better understanding of the differentiation and maturation process, we employed a standardized protocol to differentiate six hPSC lines into hepatocytes and investigated the synchronicity of the hPSC lines by applying RT-qPCR to assess the expression of lineage-specific genes (OCT4, NANOG, T, SOX17, CXCR4, CER1, HHEX, TBX3, PROX1, HNF6, AFP, HNF4a, KRT18, ALB, AAT, and CYP3A4) which serve as markers for different stages during liver development. The data was evaluated using correlation and clustering analysis, demonstrating that the expression of these markers is highly synchronized and correlated well across all cell lines. The analysis also revealed a distribution of the markers in groups reflecting the developmental stages of hepatocytes. Functional analysis of the differentiated cells further confirmed their hepatic phenotype. Taken together, these results demonstrate, on the molecular level, the highly synchronized differentiation pattern across multiple hPSC lines. Moreover, this study provides additional understanding for future efforts to improve the functionality of hPSC-derived hepatocytes and thereby increase the value of related models.
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| 17. |
- Hosseini, Kimia, et al.
(författare)
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Differentiation of Human Embryonic Stem Cells into Neuron, Cholinergic, and Glial Cells
- 2020
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Ingår i: Stem Cells International. - : HINDAWI LTD. - 1687-9678 .- 1687-966X. ; 2020
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Tidskriftsartikel (refereegranskat)abstract
- Human embryonic stem cells (hESCs) are pluripotent cells, capable of differentiation into different cellular lineages given the opportunity. Derived from the inner cell mass of blastocysts in early embryonic development, the cell self-renewal ability makes them a great tool for regenerative medicine, and there are different protocols available for maintaining hESCs in their undifferentiated state. In addition, protocols for differentiation into functional human neural stem cells (hNSCs), which have the potential for further differentiation into various neural cell types, are available. However, many protocols are time-consuming and complex and do not always fit for purpose. In this study, we carefully combined, optimized, and developed protocols for differentiation of hESCs into adherent monolayer hNSCs over a short period of time, with the possibility of both expansion and freezing. Moreover, the method details further differentiation into neurons, cholinergic neurons, and glial cells in a simple, single step by step protocol. We performed immunocytochemistry, qPCR, and electrophysiology to examine the expression profile and characteristics of the cells to verify cell lineage. Using presented protocols, the creation of neuronal cultures, cholinergic neurons, and a mixed culture of astrocytes and oligodendrocytes can be completed within a three-week time period.
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| 18. |
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| 19. |
- Kanayama, Kengo, et al.
(författare)
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Genome-wide mapping of bivalent histone modifications in hepatic stem/progenitor cells
- 2019
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Ingår i: Stem Cells International. - : Hindawi Limited. - 1687-966X .- 1687-9678. ; 2019
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Tidskriftsartikel (refereegranskat)abstract
- The “bivalent domain,” a distinctive histone modification signature, is characterized by repressive trimethylation of histone H3 at lysine 27 (H3K27me3) and active trimethylation of histone H3 at lysine 4 (H3K4me3) marks. Maintenance and dynamic resolution of these histone marks play important roles in regulating differentiation processes in various stem cell systems. However, little is known regarding their roles in hepatic stem/progenitor cells. In the present study, we conducted the chromatin immunoprecipitation (ChIP) assay followed by high-throughput DNA sequencing (ChIP-seq) analyses in purified delta-like 1 protein (Dlk + ) hepatic stem/progenitor cells and successfully identified 562 genes exhibiting bivalent domains within 2 kb of the transcription start site. Gene ontology analysis revealed that these genes were enriched in developmental functions and differentiation processes. Microarray analyses indicated that many of these genes exhibited derepression after differentiation toward hepatocyte and cholangiocyte lineages. Among these, 72 genes, including Cdkn2a and Sox4, were significantly upregulated after differentiation toward hepatocyte or cholangiocyte lineages. Knockdown of Sox4 in Dlk + cells suppressed colony propagation and resulted in increased numbers of albumin + /cytokeratin 7 + progenitor cells in colonies. These findings implicate that derepression of Sox4 expression is required to induce normal differentiation processes. In conclusion, combined ChIP-seq and microarray analyses successfully identified bivalent genes. Functional analyses of these genes will help elucidate the epigenetic machinery underlying the terminal differentiation of hepatic stem/progenitor cells.
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| 20. |
- Khatlani, T, et al.
(författare)
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Preconditioning by Hydrogen Peroxide Enhances Multiple Properties of Human Decidua Basalis Mesenchymal Stem/Multipotent Stromal Cells
- 2018
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Ingår i: Stem cells international. - : Hindawi Limited. - 1687-966X .- 1687-9678. ; 2018, s. 6480793-
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Tidskriftsartikel (refereegranskat)abstract
- Stem cell-based therapies rely on stem cell ability to repair in an oxidative stress environment. Preconditioning of mesenchymal stem cells (MSCs) to a stress environment has beneficial effects on their ability to repair injured tissues. We previously reported that MSCs from thedecidua basalis(DBMSCs) of human placenta have many important cellular functions that make them potentially useful for cell-based therapies. Here, we studied the effect of DBMSC preconditioning to a stress environment. DBMSCs were exposed to various concentrations of hydrogen peroxide (H2O2), and their functions were then assessed. DBMSC expression of immune molecules after preconditioning was also determined. DBMSC preconditioning with H2O2enhanced their proliferation, colonogenicity, adhesion, and migration. In addition, DBMSCs regardless of H2O2treatment displayed antiangiogenic activity. H2O2preconditioning also increased DBMSC expression of genes that promote cellular functions and decreased the expression of genes, which have opposite effect on their functions. Preconditioning also reduced DBMSC expression of IL-1β, but had no effects on the expression of other immune molecules that promote proliferation, adhesion, and migration. These data show that DBMSCs resist a toxic environment, which adds to their potential as a candidate stem cell type for treating various diseases in hostile environments.
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