scholarly journals Generation of iPSCs from Jaw Periosteal Cells Using Self-Replicating RNA

2019 ◽  
Vol 20 (7) ◽  
pp. 1648 ◽  
Author(s):  
Felix Umrath ◽  
Heidrun Steinle ◽  
Marbod Weber ◽  
Hans-Peter Wendel ◽  
Siegmar Reinert ◽  
...  
Keyword(s):  
Stem Cells ◽  
Fluorescent Protein ◽  
Osteogenic Potential ◽  
Safety Standards ◽  
Stem Cell Source ◽  
Periosteal Cells ◽  
Induced Pluripotent ◽  
Green Fluorescent ◽  
First Time

Jaw periosteal cells (JPCs) represent a suitable stem cell source for bone tissue engineering (BTE) applications. However, challenges associated with limited cell numbers, stressful cell sorting, or the occurrence of cell senescence during in vitro passaging and the associated insufficient osteogenic potential in vitro of JPCs and other mesenchymal stem/stromal cells (MSCs) are main hurdles and still need to be solved. In this study, for the first time, induced pluripotent stem cells (iPSCs) were generated from human JPCs to open up a new source of stem cells for BTE. For this purpose, a non-integrating self-replicating RNA (srRNA) encoding reprogramming factors and green fluorescent protein (GFP) as a reporter was used to obtain JPC-iPSCs with a feeder- and xeno-free reprogramming protocol to meet the highest safety standards for future clinical applications. Furthermore, to analyze the potential of these iPSCs as a source of osteogenic progenitor cells, JPC-iPSCs were differentiated into iPSC-derived mesenchymal stem/stromal like cells (iMSCs) and further differentiated to the osteogenic lineage under xeno-free conditions. The produced iMSCs displayed MSC marker expression and morphology as well as strong mineralization during osteogenic differentiation.

scholarly journals Could Cells from Your Nose Fix Your Heart? Transplantation of Olfactory Stem Cells in a Rat Model of Cardiac Infarction

2010 ◽  
Vol 10 ◽  
pp. 422-433 ◽  
Author(s):  
Cameron McDonald ◽  
Alan Mackay-Sim ◽  
Denis Crane ◽  
Wayne Murrell
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Fluorescent Protein ◽  
Genetically Engineered ◽  
Olfactory Mucosa ◽  
Cell Transplant ◽  
Adult Rat ◽  
Cardiac Ventricle ◽  
Green Fluorescent

This study examines the hypothesis that multipotent olfactory mucosal stem cells could provide a basis for the development of autologous cell transplant therapy for the treatment of heart attack. In humans, these cells are easily obtained by simple biopsy. Neural stem cells from the olfactory mucosa are multipotent, with the capacity to differentiate into developmental fates other than neurons and glia, with evidence of cardiomyocyte differentiationin vitroand after transplantation into the chick embryo. Olfactory stem cells were grown from rat olfactory mucosa. These cells are propagated as neurosphere cultures, similar to other neural stem cells. Olfactory neurospheres were grownin vitro, dissociated into single cell suspensions, and transplanted into the infarcted hearts of congeneic rats. Transplanted cells were genetically engineered to express green fluorescent protein (GFP) in order to allow them to be identified after transplantation. Functional assessment was attempted using echocardiography in three groups of rats: control, unoperated; infarct only; infarcted and transplanted. Transplantation of neurosphere-derived cells from adult rat olfactory mucosa appeared to restore heart rate with other trends towards improvement in other measures of ventricular function indicated. Importantly, donor-derived cells engrafted in the transplanted cardiac ventricle and expressed cardiac contractile proteins.

scholarly journals Hypoxic preconditioning enhances bone marrow mesenchymal stem cell migration via Kv2.1 channel and FAK activation

2011 ◽  
Vol 301 (2) ◽  
pp. C362-C372 ◽  
Author(s):  
Xinyang Hu ◽  
Ling Wei ◽  
Tammi M. Taylor ◽  
Jianfeng Wei ◽  
Xin Zhou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Fluorescent Protein ◽  
Hypoxic Preconditioning ◽  
Delayed Rectifier ◽  
Marrow Mesenchymal Stem Cells ◽  
Injured Region ◽  
Green Fluorescent

Transplantation using stem cells including bone marrow mesenchymal stem cells (BMSCs) is emerging as a potential regenerative therapy after ischemic attacks in the heart and brain. The migration capability of transplanted cells is a critical cellular function for tissue repair. Based on our recent observations that hypoxic preconditioning (HP) has multiple benefits in improving stem cell therapy and that the potassium Kv2.1 channel acts as a promoter for focal adhesion kinase (FAK) activation and cell motility, the present investigation tested the hypothesis that HP treatment can increase BMSC migration via the mechanism of increased Kv2.1 expression and FAK activities. BMSCs derived from green fluorescent protein-transgenic mice were treated under either normoxic (N-BMSC) or hypoxic (0.5% O2) (HP-BMSC) conditions for 24 h. Western blot analysis showed HP selectively upregulated Kv2.1 expression while leaving other K+ channels, such as Kv1.5 and Kv1.4, unaffected. Compared with normoxic controls, significantly larger outward delayed rectifier K+ currents were recorded in HP-BMSCs. HP enhanced BMSC migration/homing activities in vitro and after intravenous transplantation into rats subjected to permanent myocardial infarction (MI). The HP-promoted BMSC migration was inhibited by either blocking K+ channels or knocking down Kv2.1. Supporting a relationship among HP, Kv2.1, and FAK activation, HP increased phosphorylation of FAK397 and FAK576/577, and this effect was antagonized by blocking K+ channels. These findings provide novel evidence that HP enhances the ability of BMSCs to migrate and home to the injured region; this effect is mediated through a regulatory role of Kv2.1 on FAK phosphorylation/activation.

scholarly journals Monitoring disulfide bond formation in the eukaryotic cytosol

2004 ◽  
Vol 166 (3) ◽  
pp. 337-345 ◽  
Author(s):  
Henrik Østergaard ◽  
Christine Tachibana ◽  
Jakob R. Winther
Keyword(s):  
Fluorescent Protein ◽  
Redox Status ◽  
Reduced And Oxidized Glutathione ◽  
Cytosolic Glutathione ◽  
Green Fluorescent ◽  
Genetically Manipulated ◽  
First Time ◽  
Glutathione Redox

Glutathione is the most abundant low molecular weight thiol in the eukaryotic cytosol. The compartment-specific ratio and absolute concentrations of reduced and oxidized glutathione (GSH and GSSG, respectively) are, however, not easily determined. Here, we present a glutathione-specific green fluorescent protein–based redox probe termed redox sensitive YFP (rxYFP). Using yeast with genetically manipulated GSSG levels, we find that rxYFP equilibrates with the cytosolic glutathione redox buffer. Furthermore, in vivo and in vitro data show the equilibration to be catalyzed by glutaredoxins and that conditions of high intracellular GSSG confer to these a new role as dithiol oxidases. For the first time a genetically encoded probe is used to determine the redox potential specifically of cytosolic glutathione. We find it to be −289 mV, indicating that the glutathione redox status is highly reducing and corresponds to a cytosolic GSSG level in the low micromolar range. Even under these conditions a significant fraction of rxYFP is oxidized.

Effect of MicroRNA-20a on Osteogenic Differentiation of Human Adipose Tissue-Derived Stem Cells

2019 ◽  
Vol 208 (3-4) ◽  
pp. 148-157
Author(s):  
Tao Luo ◽  
Xueqin Yang ◽  
Yan Sun ◽  
Xinqi Huang ◽  
Ling Zou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Adipose Tissue ◽  
Osteogenic Differentiation ◽  
Fluorescent Protein ◽  
Mrna Level ◽  
Human Adipose Tissue ◽  
Associated Proteins ◽  
Green Fluorescent

Osteogenic differentiation of human adipose tissue-derived stem cells (hASCs) is a complex process that is regulated by multiple factors, including microRNAs (miRNAs). The miRNA miR-20a was shown to promote bone formation from bone marrow-derived mesenchymal stem cells. However, the role of miR-20a in osteogenic differentiation of hASCs remains unclear. In this study, we systematically evaluated the function of miR-20a in regulating hASC osteogenesis in vitro. hASCs were transduced with miR-20a-overexpressing and miR-20a-sponge lentiviral vectors, with green fluorescent protein (GFP) as a control. The results showed that miR-20a transcription was upregulated after hASC mineralization. Compared with the miR-20a-sponge, GFP, and hASC groups, the miR-20a-overexpressing group showed higher alkaline phosphatase (ALP) activity on days 7 and 14. Moreover, the mRNA level of ALP increased significantly in the miR-20a-overexpressing group on day 14. Furthermore, the protein of the target gene PPARγ was decreased, and the osteogenic differentiation-associated proteins ALP, osteocalcin, and RUNX2 were upregulated. hASCs anchored to HA/β-TCP revealed a healthy polygonal morphology and developed cytoplasmic extensions. miR-20a promoted osteogenic differentiation of the cell scaffold. Taken together, these data ­confirm that miRNA-20a promotes the osteogenesis of hASCs in vitro, and its essential role in vivo needs further ­investigation.

scholarly journals An Efficient Intestinal Organoid System of Direct Sorting to Evaluate Stem Cell Competition in Vitro

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Yuki Fujimichi ◽  
Kensuke Otsuka ◽  
Masanori Tomita ◽  
Toshiyasu Iwasaki
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Fluorescent Protein ◽  
Lineage Tracing ◽  
Cell Competition ◽  
Quality Control Mechanism ◽  
Green Fluorescent ◽  
G Protein Coupled ◽  
Culture Protocol

AbstractStem cell competition could shed light on the tissue-based quality control mechanism that prevents carcinogenesis. To quantitatively evaluate stem cell competition in vitro, we developed a two-color intestinal organoid forming system. First, we improved a protocol of culturing organoids from intestinal leucine-rich-repeat containing G-protein-coupled receptor 5 (Lgr5)- enhanced green fluorescent protein (EGFP)high stem cells directly sorted on Matrigel without embedding. The organoid-forming potential (OFP) was 25% of Lgr5-EGFPhigh cells sorted at one cell per well. Using this culture protocol with lineage tracing, we established a two-color organoid culture system by mixing stem cells expressing different fluorescent colors. To analyze stem cell competition, two-color organoids were formed by mixing X-ray-irradiated and non-irradiated intestinal stem cells. In the two-color organoids, irradiated stem cells exhibited a growth disadvantage, although the OFP of irradiated cells alone did not decrease significantly from that of non-irradiated cells. These results suggest that stem cell competition can be evaluated quantitively in vitro using our new system.

Partial Reprogramming As An Emerging Strategy for Safe Induced Cell Generation and Rejuvenation

2019 ◽  
Vol 19 (4) ◽  
pp. 248-254
Author(s):  
Marianne Lehmann ◽  
Martina Canatelli-Mallat ◽  
Priscila Chiavellini ◽  
Gloria M. Cónsole ◽  
Maria D. Gallardo ◽  
...  
Keyword(s):  
Stem Cells ◽  
Somatic Cell ◽  
Fluorescent Protein ◽  
Somatic Cells ◽  
Cell Types ◽  
Cell Reprogramming ◽  
Green Fluorescent ◽  
Original Cell

Background: Conventional cell reprogramming involves converting a somatic cell line into induced pluripotent stem cells (iPSC), which subsequently can be re-differentiated to specific somatic cell types. Alternatively, partial cell reprogramming converts somatic cells into other somatic cell types by transient expression of pluripotency genes thus generating intermediates that retain their original cell identity, but are responsive to appropriate cocktails of specific differentiation factors. Additionally, biological rejuvenation by partial cell reprogramming is an emerging avenue of research. Objective: Here, we will briefly review the emerging information pointing to partial reprogramming as a suitable strategy to achieve cell reprogramming and rejuvenation, bypassing cell dedifferentiation. Methods: In this context, regulatable pluripotency gene expression systems are the most widely used at present to implement partial cell reprogramming. For instance, we have constructed a regulatable bidirectional adenovector expressing Green Fluorescent Protein and oct4, sox2, klf4 and c-myc genes (known as the Yamanaka genes or OSKM). Results: Partial cell reprogramming has been used to reprogram fibroblasts to cardiomyocytes, neural progenitors and neural stem cells. Rejuvenation by cyclic partial reprogramming has been achieved both in vivo and in cell culture using transgenic mice and cells expressing the OSKM genes, respectively, controlled by a regulatable promoter. Conclusion: Partial reprogramming emerges as a powerful tool for the genesis of iPSC-free induced somatic cells of therapeutic value and for the implementation of in vitro and in vivo rejuvenation keeping cell type identity unchanged.

Characterisation of the deleted in azoospermia like (Dazl)–green fluorescent protein mouse model generated by a two-step embryonic stem cell-based strategy to identify pluripotent and germ cells

2016 ◽  
Vol 28 (11) ◽  
pp. 1741 ◽  
Author(s):  
Priscila Ramos-Ibeas ◽  
Eva Pericuesta ◽  
Raúl Fernández-González ◽  
Alfonso Gutiérrez-Adán ◽  
Miguel Ángel Ramírez
Keyword(s):  
Stem Cells ◽  
Green Fluorescent Protein ◽  
Transgenic Mice ◽  
Germ Cells ◽  
Fluorescent Protein ◽  
Preimplantation Embryo ◽  
Embryonic Stem ◽  
Deleted In Azoospermia ◽  
Green Fluorescent

The deleted in azoospermia like (Dazl) gene is preferentially expressed in germ cells; however, recent studies indicate that it may have pluripotency-related functions. We generated Dazl–green fluorescent protein (GFP) transgenic mice and assayed the ability of Dazl-driven GFP to mark preimplantation embryo development, fetal, neonatal and adult tissues, and in vitro differentiation from embryonic stem cells (ESCs) to embryoid bodies (EBs) and to primordial germ cell (PGC)-like cells. The Dazl-GFP mice were generated by a two-step ESC-based strategy, which enabled primary and secondary screening of stably transfected clones before embryo injection. During preimplantation embryo stages, GFP was detected from the zygote to blastocyst stage. At Embryonic Day (E) 12.5, GFP was expressed in gonadal ridges and in neonatal gonads of both sexes. In adult mice, GFP expression was found during spermatogenesis from spermatogonia to elongating spermatids and in the cytoplasm of oocytes. However, GFP mRNA was also detected in other tissues harbouring multipotent cells, such as the intestine and bone marrow. Fluorescence was maintained along in vitro Dazl-GFP ESC differentiation to EBs, and in PGC-like cells. In addition to its largely known function in germ cell development, Dazl could have an additional role in pluripotency, supporting these transgenic mice as a valuable tool for the prospective identification of stem cells from several tissues.

scholarly journals Isolation, Characterization, and Transduction of Endometrial Decidual Tissue Multipotent Mesenchymal Stromal/Stem Cells from Menstrual Blood

2013 ◽  
Vol 2013 ◽  
pp. 1-14 ◽  
Author(s):  
Filippo Rossignoli ◽  
Anna Caselli ◽  
Giulia Grisendi ◽  
Serena Piccinno ◽  
Jorge S. Burns ◽  
...  
Keyword(s):  
Stem Cells ◽  
Fluorescent Protein ◽  
Menstrual Blood ◽  
Decidual Tissue ◽  
Promising Tool ◽  
Clonogenic Potential ◽  
Cell And Gene Therapy ◽  
Green Fluorescent ◽  
Stromal Stem Cells

Mesenchymal stromal/stem cells (MSCs) reveal progenitor cells-like features including proliferation and differentiation capacities. One of the most historically recognized sources of MSC has been the bone marrow, while other sources recently include adipose tissue, teeth, bone, muscle, placenta, liver, pancreas, umbilical cord, and cord blood. Frequently, progenitor isolation requires traumatic procedures that are poorly feasible and associated with patient discomfort. In the attempt to identify a more approachable MSC source, we focused on endometrial decidual tissue (EDT) found within menstrual blood. Based also on recent literature findings, we hypothesized that EDT may contain heterogeneous populations including some having MSC-like features. Thus, we here sought to isolate EDT-MSC processing menstrual samples from multiple donors. Cytofluorimetric analyses revealed that resulting adherent cells were expressing mesenchymal surface markers, including CD56, CD73, CD90, CD105 and CD146, and pluripotency markers such as SSEA-4. Moreover, EDT-MSC showed a robust clonogenic potential and could be largely expandedin vitroas fibroblastoid elements. In addition, differentiation assays drove these cells towards osteogenic, adipogenic, and chondrogenic lineages. Finally, for the first time, we were able to gene modify these progenitors by a retroviral vector carrying the green fluorescent protein. From these data, we suggest that EDT-MSC could represent a new promising tool having potential within cell and gene therapy applications.

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