scholarly journals The Roll of Toll-like Receptors in the Stem Cell Inflammatory Response

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Katelyn Lawson ◽  
Troy Markel
Keyword(s):  
Stem Cells ◽  
Inflammatory Mediators ◽  
Negative Control ◽  
National Institutes Of Health ◽  
In Vivo Model ◽  
Toll Like Receptors ◽  
The Media ◽  
Air Cells ◽  
Positive Results

Background: Toll-like receptors serve as ligands for LPS and other inflammatory mediators. Previous studies have shown that TLR4 is deleterious, while TLR9 is beneficial in the setting of inflammation and ischemia. Umbilical stem cells (USCs) have shown promise in the acute treatment of inflammation but their response to inflammatory mediators has not been fully elucidated. We hypothesized that knockdown of TLR4 in USCs would result in lower levels of IL-6 and VEGF production, while knockdown of TLR9 would produce higher levels of these cytokines when cells were exposed to LPS or hypoxia.    Methods: USCs were cultured in polystyrene flasks in Mesenpro media at 37C in 5% CO2 in air. Cells were plated into 12-well plates at a concentration of 100,000 cells/well. Cells were transfected for 24h with siRNA to knockdown TLR4 and TLR9, respectively. Knockdown was confirmed by PCR. Experimental groups were: 1) Control, 2) Scramble siRNA for negative control, 3) TLR4 siRNA and 4) TLR9 siRNA. After 24 hours the media was changed and cells were exposed to either LPS (200ng/ml) or 5% oxygen for 24 hours. The supernatant was then collected and analyzed with ELISA for VEGF and IL-6. Data were analyzed by Mann Whitney test and p<0.05 was significant.     Results: TLR4 and TLR9 were effectively knocked down by the transfection process. However, no significant levels of VEGF or IL-6 were detected from any of the experimental groups.    Conclusion and Implications: Although no significant levels of VEGF or IL-6 were detected in the ELISA after exposure to inflammatory agents, there is still indication that TLR4 and TLR9 play critical roles in inflammation. The experiment should be run again and tested for more inflammatory cytokines. Positive results from this study can translate to an in vivo model, in which engineered TLR4KD stem cells have the potential to minimize tissue inflammation and beyond standard cell therapy.    Acknowledgement: This project was funded, in part, with support from the Indiana Clinical and Translational Sciences Institute funded, in part by UL1TR002529 from the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.   

197 THE EFFECT OF ZINC ON THE DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS INTO OSTEOBLASTS

2017 ◽  
Vol 29 (1) ◽  
pp. 207 ◽  
Author(s):  
J. C. Bertels ◽  
M. Rubessa ◽  
S.R. Schreiber ◽  
M. B. Wheeler
Keyword(s):  
Stem Cells ◽  
Bone Formation ◽  
Negative Control ◽  
Nodule Formation ◽  
Adipose Derived Stem Cells ◽  
Osteogenic Medium ◽  
Alizarin Red ◽  
The Media ◽  
Positive Effect

The aim of this project was to evaluate the effects of zinc in osteogenic media and its effect on the differentiation of adipose-derived stem cells (ASC) into osteoblasts. Zinc has a stimulatory effect on bone formation and mineralization in vivo and vitro (Seo et al. 2010 Nutr. Res. Pract. 4, 356–361). Our hypothesis was that the presence of zinc in the osteogenic media would positively influence both the speed of formation and the number of osteoblastic nodules formed. Swine ASC were isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20–33). The ASC were divided in 8 different treatments: 6 different concentrations of zinc in the osteogenic medium (8, 4, 0.8, 0.4, 0.08, and 0.04 mM) plus 2 control treatments (osteogenic medium without zinc and a negative control, DMEM). The media was changed twice a week for 4 weeks. The experiment was replicated 4 times. At the end of the culture period, cells were stained with Alizarin Red S. In each well, we counted the nodules and divided them in 2 categories: formed and forming nodules. The second evaluation that we did was to evaluate the diameter of the largest nodules (2/well) in each group. Data were analysed by ANOVA using the Generalized Linear Model procedure (SPSS, IBM Corp., Armonk, NY, USA). Bonferroni’s post-hoc test was used to perform statistical multiple comparison. The α-level was set at 0.05. The results showed that the doses of zinc of both 4 and 8 mM were toxic to the whole cell populations in this treatment, which was indicated by cell death, whereas the concentrations of 0.8 and 0.4 mM were not cytotoxic but no nodules formed. Here we report the results that are greater than zero in Table 1. There is a positive effect on nodule formation when the zinc is added to the media. It is clear that the total number of nodules is different between the 0.08 mM zinc group and the control (P < 0.003). When we evaluated nodule diameter we found a direct correlation between the zinc concentration and the diameter of the nodules: 292.7 (±136.6) v. 366.8 (±218.7) v. 423.7 (±267.7) µm for the control, 0.04 mM zinc, and 0.08 mM zinc, respectively. The largest nodule was found in the 0.08 mM zinc treatment at 886.6 µm. These results confirmed the positive effect of this mineral on bone formation. This preliminary experiment is the first step towards the analysis of the behaviour of ASC on scaffolds with zinc incorporated into their matrix. Table 1. The average number (SD in parentheses) of formed and forming osteoblast nodules compared between treatment groups

scholarly journals Immunosuppressants Affect Human Neural Stem Cells In Vitro but Not in an In Vivo Model of Spinal Cord Injury

2013 ◽  
Vol 2 (10) ◽  
pp. 731-744 ◽  
Author(s):  
Christopher J. Sontag ◽  
Hal X. Nguyen ◽  
Noriko Kamei ◽  
Nobuko Uchida ◽  
Aileen J. Anderson ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Stem Cells ◽  
Spinal Cord Injury ◽  
Neural Stem Cells ◽  
In Vivo Model ◽  
Human Neural Stem Cells ◽  
Cord Injury

10. Embryonic Stem Cells Used for Disc Regeneration in an In Vivo Model of Disc Degeneration

2008 ◽  
Vol 8 (5) ◽  
pp. 5S ◽  
Author(s):  
Ramiro Perez De La Torre ◽  
Hormoz Sheikh ◽  
Mick Perez-Cruet ◽  
Chistopher Fecek ◽  
Rasul Chaudhry ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Disc Degeneration ◽  
Embryonic Stem ◽  
In Vivo Model ◽  
Disc Regeneration

scholarly journals Use of 5-fluorouracil to analyze the effect of macrophage inflammatory protein-1 alpha on long-term reconstituting stem cells in vivo

Blood ◽  
1993 ◽  
Vol 81 (6) ◽  
pp. 1497-1504 ◽  
Author(s):  
VF Quesniaux ◽  
GJ Graham ◽  
I Pragnell ◽  
D Donaldson ◽  
SD Wolpe ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Inhibitory Effect ◽  
In Vivo Model ◽  
Hematopoietic Progenitors ◽  
Inflammatory Protein ◽  
Macrophage Inflammatory Protein

Abstract A macrophage-derived inhibitor of early hematopoietic progenitors (colony-forming unit-spleen, CFU-A) called stem cell inhibitor was found to be identical to macrophage inflammatory protein-1 alpha (MIP-1 alpha). We investigated the effect of MIP-1 alpha on the earliest stem cells that sustain long-term hematopoiesis in vivo in a competitive bone marrow repopulation assay. Because long-term reconstituting (LTR) stem cells are normally quiescent, an in vivo model was first developed in which they are triggered to cycle. A first 5-fluorouracil (5-FU) injection was used to eliminate later progenitors, causing the LTR stem cells, which are normally resistant to 5-FU, to enter the cell cycle and become sensitive to a second 5-FU injection administered 5 days later. Human MIP-1 alpha administered from day 0 to 7 was unable to prevent the depletion of the LTR stem cells by the second 5-FU treatment, as observed on day 7 in this model, suggesting that the LTR stem cells were not prevented from being triggered into cycle despite the MIP-1 alpha treatment. However, the MIP-1 alpha protocol used here did substantially decrease the number of more mature hematopoietic progenitors (granulocyte-macrophage colony-forming cells [CFC], burst- forming unit-erythroid, CFCmulti, and preCFCmulti) recovered in the bone marrow shortly after a single 5-FU injection. In vitro, MIP-1 alpha had no inhibitory effect on the ability of these progenitors to form colonies. This study confirms the in vivo inhibitory effect of MIP- 1 alpha on subpopulations of hematopoietic progenitors that are activated in myelodepressed animals. However, MIP-1 alpha had no effect on the long-term reconstituting stem cells in vivo under conditions in which it effectively reduced all later progenitors.

DISTINCT IMPLICATION OF BONE MARROW STEM CELLS IN TWO IN VIVO MODEL OF PATHOLOGICAL ANGIOGENESIS

2003 ◽  
Vol 13 (Suppl 1) ◽  
pp. 58.3-58
Author(s):  
M. Jost ◽  
V. Lambert ◽  
C. Maillard ◽  
K. Bajou ◽  
C. Humblet ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Stem Cells ◽  
In Vivo Model ◽  
Pathological Angiogenesis

scholarly journals Modulation of Adult Mesenchymal Stem Cells Activity by Toll-Like Receptors: Implications on Therapeutic Potential

2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Olga DelaRosa ◽  
Eleuterio Lombardo
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Therapeutic Potential ◽  
Inflammatory Diseases ◽  
Culture Conditions ◽  
Toll Like Receptors ◽  
Adult Mesenchymal Stem Cells ◽  
Special Relevance ◽  
Tlr Signalling

Mesenchymal stem cells (MSCs) are of special interest as therapeutic agents in the settings of both chronic inflammatory and autoimmune diseases. Toll-like receptors (TLR) ligands have been linked with the perpetuation of inflammation in a number of chronic inflammatory diseases due to the permanent exposure of the immune system to TLR-specific stimuli. Therefore, MSCs employed in therapy can be potentially exposed to TLR ligands, which may modulate MSC therapeutic potential in vivo. Recent results demonstrate that MSCs are activated by TLR ligands leading to modulation of the differentiation, migration, proliferation, survival, and immunosuppression capacities. However inconsistent results among authors have been reported suggesting that the source of MSCs, TLR stimuli employed or culture conditions play a role. Notably, activation by TLR ligands has not been reported to modulate the “immunoprivileged” phenotype of MSCs which is of special relevance regarding the use of allogeneic MSC-based therapies. In this review, we discuss the available data on the modulation of MSCs activity through TLR signalling.

Xenotransplantation of Primary CD34+CD38- Hematopoietic Stem Cells Results in an In Vivo Model of Idiopathic Myelofibrosis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 666-666
Author(s):  
Noriyuki Saito ◽  
Fumihiko Ishikawa ◽  
Kazuya Shimoda ◽  
Shuro Yoshida ◽  
Yoriko Saito ◽  
...  
Keyword(s):  
Stem Cells ◽  
In Vivo Model ◽  
Idiopathic Myelofibrosis ◽  
Hematopoietic Stem ◽  
Hematopoietic Reconstitution ◽  
Cd34 Cells ◽  
Normal Human ◽  
Myelomonocytic Cells ◽  
Xenotransplantation Model

Abstract Idiopathic myelofibrosis (IMF) is characterized by clonal proliferation of abnormal myelomonocytic cells and megakaryocytes. These abnormal cells secrete various cytokines resulting in reactive fibrosis and increased collagen content in the bone marrow (BM), and lead to extramedullary hematopoiesis and the appearance of CD34+ cells in the peripheral blood (PB). Although IMF is thought to originate at the level of hematopoietic stem cell (HSC), this has not been demonstrated directly in primary human IMF. To demonstrate the involvement of HSCs in the pathogenesis of IMF and to establish an in vivo model of IMF, we used the newborn NOD/SCID/IL2rg-null xenotransplantation model. We purified PB CD34+ cells from six IMF patients, transplanted 1–10 x10e4 cells intravenously into newborn NOD/SCID/IL2rg-null recipients and analyzed PB and BM human CD45+ hematopoietic cell chimerism, degree of suppression of murine hematopoiesis, presence of hallmark BM fibrosis and plasma TGF-b1 levels in the recipients at 6 months post-transplantation. Primary IMF PB CD34+ cells from five out of six patients engrafted in twelve out of twelve recipients. BM of all engrafted recipients demonstrated fibrotic changes associated with increased proliferation of murine fibroblasts, the presence of human megakaryocytes and elevated plasma TGF-b1 levels, recapitulating the clinical features of IMF. Three distinct patterns of human hematopoietic reconstitution were observed among the engrafted recipients: Predominantly malignant myelomonocytic engraftment in the PB and BM (n=4), Reconstitution of both normal human hematopoiesis (with mature B and T cells, myeloid cells and platelets) and malignant myelomonocytic cells (n=6) and Development of acute leukemia (n=2). Fibrotic change was seen even in the BM of recipients that showed normal human hematopoietic reconstitution, showing that in IMF, there is co-existence of both normal and malignant hematopoietic stem/progenitor cells in the PB CD34+ fraction. Furthermore, when 5–10 x 10e3 sorted PB CD34+CD38– cells from three patients were transplanted into six newborn NOD/SCID/IL2rg-null recipients, reconstitution with human myelomonocytic cells associated with BM fibrosis was demonstrated in all recipients, with compatible level of PB and BM chimerism with those transplanted with PB CD34+ cells. These findings demonstrate that the IMF-initiating cells are contained within the HSC fraction. The newborn NOD/SCID/IL2rg-null xenotransplantation model provides an in vivo model of primary human IMF that may lead to better understanding of the mechanisms of IMF pathogenesis including the identification of IMF stem cells and may be useful for development of novel therapeutic agents for IMF.

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