scholarly journals Derivation and Characterization of EGFP-Labeled Rabbit Limbal Mesenchymal Stem Cells and Their Potential for Research in Regenerative Ophthalmology

Biomedicines ◽  
2021 ◽  
Vol 9 (9) ◽  
pp. 1134
Author(s):  
Julia I. Khorolskaya ◽  
Daria A. Perepletchikova ◽  
Daniel V. Kachkin ◽  
Kirill E. Zhurenkov ◽  
Elga I. Alexander-Sinkler ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Fluorescent Protein ◽  
Rabbit Model ◽  
Stem Cell Markers ◽  
Epithelial Stem Cells ◽  
Limbal Stem Cell ◽  
Green Fluorescent

The development of cell-based approaches to the treatment of various cornea pathologies, including limbal stem cell deficiency (LSCD), is an area of current interest in regenerative biomedicine. In this context, the shortage of donor material is urgent, and limbal mesenchymal stem cells (L-MSCs) may become a promising cell source for the development of these novel approaches, being established mainly within the rabbit model. In this study, we obtained and characterized rabbit L-MSCs and modified them with lentiviral transduction to express the green fluorescent protein EGFP (L-MSCs-EGFP). L-MSCs and L-MSCs-EGFP express not only stem cell markers specific for mesenchymal stem cells but also ABCG2, ABCB5, ALDH3A1, PAX6, and p63a specific for limbal epithelial stem cells (LESCs), as well as various cytokeratins (3/12, 15, 19). L-MSCs-EGFP have been proven to differentiate into adipogenic, osteogenic, and chondrogenic directions, as well as to transdifferentiate into epithelial cells. The possibility of using L-MSCs-EGFP to study the biocompatibility of various scaffolds developed to treat corneal pathologies was demonstrated. L-MSCs-EGFP may become a useful tool for studying regenerative processes occurring during the treatment of various corneal pathologies, including LSCD, with the use of cell-based technologies.

Phenotypic and Molecular Characterization of Bone Marrow L-NGFR + Mesenchymal Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2333-2333 ◽  
Author(s):  
Nadia Quirici ◽  
Davide Soligo ◽  
Chiara Borsotti ◽  
Cinzia Scavullo ◽  
Stefano Zangrossi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Conditioned Media ◽  
Cell Phenotype ◽  
Stem Cell Markers ◽  
Multilineage Differentiation ◽  
Hematopoietic Stem

Abstract In a previous report we demonstrated that the immunomagnetic sorting of bone marrow (BM) cells labeled with low-affinity nerve growth factor receptor (L-NGFR) antibodies allows the selection of phenotypically and functionally homogeneous cells that are capable of expansion, self-renewal and differentiation into multiple mesenchymal cells lineages. Furthermore, we reported the presence of a subpopulation of L-NGFR+ cells coexpressing CD133 and CD34, markers associated with a primitive hematopoietic stem cell phenotype. In the present study we expanded on the phenotypic characterization of these cells and investigated their potential for multilineage differentiation. BM L-NGFR+ cells were analyzed by flow cytometry immediately after immunoseparation and the expression of a variety of stem cell markers was studied. In 12 subsequent experiments L-NGFR+ cells expressed CD45low (97.5% ±3), CD34 (19.9%±13), CD133 (10.4%±6), CD105 (46.8%±36%), P1H12 (50.5%±18), KDR (34%±18) and SSEA-3 (0.47%±0.41). In addition L-NGFR+ expressed high levels of the SCF ligand CD117 (40%±16%). As we previously demonstrated, L-NGFR antibodies identify a subpopulation of cells with a high proliferative capacity and potential for multilineage differentiation along the mesenchymal lineage. We now show, in accordance to these phenotypic data, that the L-NGFR+ cells in the presence of SCF (100 ng/ml) doubled the number of CFU-F and expanded both adipocytic and osteoblastic differentiation in comparison to mesenchymal cultures without growth factors or supplemented with Flt-3L+IL-6 (both 100 ng/ml). SCF seems therefore to act at least as a survival/proliferation factor for mesenchymal stem cells. Transdifferentiation potentialities towards endothelium were determined incubating L-NGFR+ cells in M199 supplemented with 10% FBS, 50 ng/ml VEGF, 1 ng/ml bFGF and 2 ng/ml IGF-1. At confluence, the cells were further purified using Ulex europaeus agglutinin-1 (UEA-1)-fluorescein isothiocyanate (FITC) and anti-FITC microbeads and expanded with VEGF. Immunophenotypic analysis of 8 samples showed a variable expression of endothelial markers: P1H12 ranging from 17 to 58%, CD105 from 98–100% and CD202b from 18 to 100%. L-NGFR+ cells, immediately after immunoseparation, were expressing Desmin but not MyoD, Miogenin, Mrf4, Myf5 by means of RT-PCR, while these cells were expressing NSE, TRKA and GalC, but not Nestin and GFAP. Experiments are ongoing to demonstrate muscle and neuron-glial differentiation in vitro using specific media (DMEM 10% FBS + 3 mM %-azacytidine, astrocyte conditioned media, neural stem cell conditioned media), In conclusion, the expression on NGFR+ cells of a variety of markers, not exclusively related to the mesenchymal lineage, and the reproducible ability to differentiate endothelial cells suggest that these cells may represent a subset of adult MSC with some multipotentiality.

Distribution of amniotic stem cells in human term amnion membrane

Microscopy ◽  
2021 ◽  
Author(s):  
Nobuyuki Koike ◽  
Jun Sugimoto ◽  
Motonori Okabe ◽  
Kenichi Arai ◽  
Makiko Nogami ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Stem Cell Markers ◽  
Epithelial Stem Cells ◽  
Human Amnion ◽  
Transporter Protein ◽  
Amniotic Mesenchymal Stem Cells ◽  
Amnion Membrane ◽  
And Function ◽  
A Site

Abstract Amnion membrane studies related to miscarriage have been conducted in the field of obstetrics and gynecology. However, the distribution of stem cells within the amnion and the differences in the properties of each type of stem cells are still not well understood. We address this gap in knowledge in the present study where we morphologically classified the amnion membrane, and we clarified the distribution of stem cells here to identify functionally different amniotic membrane–derived stem cells. The amnion can be divided into a site that is continuous with the umbilical cord (region A), a site that adheres to the placenta (region B), and a site that is located opposite the placenta (region C). We found that human amnion epithelial stem cells (HAECs) that strongly express stem cell markers were abundant in area A. HAEC not only expressesed stem cell-specific surface markers TRA-1-60, Tra-1-81, SSEA4, SSEA3, but was also OCT-3/4 positive and had alkaline phosphatase activity. Human amniotic mesenchymal stem cells expressed KLF-A, OCTA, Oct3/4, c-MYC and Sox2 which is transcription factor. Especially, in regions A and B they have expressed CD73, and the higher expression of BCRP which is drug excretion transporter protein than the other parts. These data suggest that different types of stem cells may have existed in different area. The understanding the relation with characteristics of the stem cells in each area and function would allow for the efficient harvest of suitable HAE and HAM stem cells as using tool for regenerative medicine.

Heterogeneity in histone 2B-green fluorescent protein-retaining putative small intestinal stem cells at cell position 4 and their absence in the colon

2012 ◽  
Vol 303 (11) ◽  
pp. G1188-G1201 ◽  
Author(s):  
Kevin R. Hughes ◽  
Ricardo M. C. Gândara ◽  
Tanvi Javkar ◽  
Fred Sablitzky ◽  
Hanno Hock ◽  
...  
Keyword(s):  
Stem Cells ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Paneth Cells ◽  
Epithelial Stem Cells ◽  
Epithelial Regeneration ◽  
Cell Position ◽  
Small Intestinal ◽  
Green Fluorescent ◽  
Dose Radiation

Stem cells have been identified in two locations in small intestinal crypts; those intercalated between Paneth cells and another population (which retains DNA label) are located above the Paneth cell zone, at cell position 4. Because of disadvantages associated with the use of DNA label, doxycycline-induced transient transgenic expression of histone 2B (H2B)-green fluorescent protein (GFP) was investigated. H2B-GFP-retaining putative stem cells were consistently seen, with a peak at cell position 4, over chase periods of up to 112 days. After a 28-day chase, a subpopulation of the H2B-GFP-retaining cells was cycling, but the slow cycling status of the majority was illustrated by lack of expression of pHistone H3 and Ki67. Although some H2B-GFP-retaining cells were sensitive to low-dose radiation, the majority was resistant to low- and high-dose radiation-induced cell death, and a proportion of the surviving cells proliferated during subsequent epithelial regeneration. Long-term retention of H2B-GFP in a subpopulation of small intestinal Paneth cells was also seen, implying that they are long lived. In contrast to the small intestine, H2B-GFP-retaining epithelial cells were not seen in the colon from 28-day chase onward. This implies important differences in stem cell function between these two regions of the gastrointestinal tract, which may have implications for region-specific susceptibility to diseases (such as cancer and ulcerative colitis), in which epithelial stem cells and their progeny are involved.

Isolation and Characterization of Human Epidermal Stem Cells

1996 ◽  
Vol 91 (2) ◽  
pp. 141-146 ◽  
Author(s):  
P. H. Jones
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Matrix Proteins ◽  
Stem Cell Markers ◽  
Epidermal Stem Cells ◽  
Cell Behaviour ◽  
Isolation And Characterization ◽  
Integrin Family

1. The keratinocytes in human epidermis are constantly turned over and replaced by a population of stem cells located in the basal epidermal layer. Until recently there were no markers allowing the isolation of viable epidermal stem cells. However, it has now been shown that epidermal stem cells can be isolated both in vitro and direct from the epidermis as they express high levels of functional β1 integrin family receptors for extracellular matrix proteins. 2. The evidence for integrins as stem cell markers and the insights that have been gained into stem cell behaviour are reviewed.

Stem Cell Defect in Ubiquitin-Green Fluorescent Protein Mice Facilitates Engraftment of Lymphoid-Primed Hematopoietic Stem Cells

Stem Cells ◽  
2018 ◽  
Vol 36 (8) ◽  
pp. 1237-1248
Author(s):  
Kateřina Faltusová ◽  
Katarína Szikszai ◽  
Martin Molík ◽  
Jana Linhartová ◽  
Petr Páral ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Green Fluorescent Protein ◽  
Hematopoietic Stem Cells ◽  
Fluorescent Protein ◽  
Hematopoietic Stem ◽  
Green Fluorescent ◽  
Cell Defect

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.

Sign in / Sign up

Export Citation Format

Share Document