Alterations of epigenetic regulators and P53 mutations in murine mesenchymal stem cell cultures: A possible mechanism of spontaneous transformation

2021 ◽  
pp. 1-11
Author(s):  
Ayman Mohamed Metwally ◽  
Hanchen Li ◽  
Jean Marie Houghton
Keyword(s):  
Stromal Cells ◽  
P53 Mutation ◽  
Dna Binding Domain ◽  
P53 Mutations ◽  
Spontaneous Transformation ◽  
The Third ◽  
Culture Model ◽  
Epigenetic Regulators ◽  
Transformation Methods

BACKGROUND: Recent studies demonstrated the involvement of mesenchymal stem/stromal cells (MSCs) in carcinogenesis, but the molecular mechanism behind this transformation is still obscured. OBJECTIVE: To screen both the expression levels of polycomb and trithorax epigenetic regulators and TrP53 mutations in early and late MSC culture passages in an attempt to decipher the mechanism of spontaneous transformation. METHODS: The study was conducted on early and late passages of MSC culture model from C57BL/6J mice. The expression profile of 84 epigenetic regulators was examined using RT2 profiler PCR array. TrP53 mutations in the DNA binding domain was screened. Codons, amino acids positions and the corresponding human variants were detected in P53 sequences. RESULTS: Sixty-two epigenetic regulators were dysregulated. Abnormalities were detected starting the third passage. Nine regulators were dysregulated in all passages. (C>G) substitution P53 mutation was detected in passage 3 resulting in Ser152Arg substitution. Passages 6, 9, 12 and the last passage showed T>C substitution resulting in Cys235Arg substitution. The last passage had T deletion and A insertion resulting in frame shift mutations changing the p.Phe286Ser and p.Asn103Lys respectively. CONCLUSION: In vitro expanded MSCs undergo transformation through alteration of epigenetic regulators which results in genomic instability and frequent P53 mutations.

scholarly journals Endometrial stromal cells regulate epithelial cell growth in vitro: a new co-culture model

2001 ◽  
Vol 16 (5) ◽  
pp. 836-845 ◽  
Author(s):  
Julia T. Arnold ◽  
David G. Kaufman ◽  
Markku Seppälä ◽  
Bruce A. Lessey
Keyword(s):  
Cell Growth ◽  
Epithelial Cell ◽  
Stromal Cells ◽  
Endometrial Stromal Cells ◽  
Culture Model

scholarly journals Sex- and Mutation-Specific p53 Gain-of-Function Activity in Gliomagenesis

2021 ◽  
Vol 1 (3) ◽  
pp. 148-163
Author(s):  
Nathan C. Rockwell ◽  
Wei Yang ◽  
Nicole M. Warrington ◽  
Max V. Staller ◽  
Malachi Griffith ◽  
...  
Keyword(s):  
Sex Differences ◽  
Dna Binding ◽  
Sex Difference ◽  
P53 Mutation ◽  
Mutant P53 ◽  
Missense Mutations ◽  
P53 Mutations ◽  
Primary Mouse

In cancer, missense mutations in the DNA-binding domain of TP53 are common. They abrogate canonical p53 activity and frequently confer gain-of-oncogenic function (GOF) through localization of transcriptionally active mutant p53 to noncanonical genes. We found that several recurring p53 mutations exhibit a sex difference in frequency in patients with glioblastoma (GBM). In vitro and in vivo analysis of three mutations, p53R172H, p53Y202C, and p53Y217C, revealed unique interactions between cellular sex and p53 GOF mutations that determined each mutation's ability to transform male versus female primary mouse astrocytes. These phenotypic differences were correlated with sex- and p53 mutation–specific patterns of genomic localization to the transcriptional start sites of upregulated genes belonging to core cancer pathways. The promoter regions of these genes exhibited a sex difference in enrichment for different transcription factor DNA-binding motifs. Together, our data establish a novel mechanism for sex-specific mutant p53 GOF activity in GBM with implications for all cancer. Significance: Sex differences in cancer, including glioblastoma, have been observed in both incidence and outcome. We reveal that TP53, the most commonly mutated gene in cancer, contributes to sex differences through differential GOF activity. This discovery has critical implications for our understanding of p53 mutations and the importance of sex as a biological variable.

Effect of adipose-derived mesenchymal stromal cells on tendon healing in aging and estrogen deficiency: an in vitro co-culture model

Cytotherapy ◽  
2015 ◽  
Vol 17 (11) ◽  
pp. 1536-1544 ◽  
Author(s):  
Francesca Veronesi ◽  
Elena Della Bella ◽  
Paola Torricelli ◽  
Stefania Pagani ◽  
Milena Fini
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Tendon Healing ◽  
Estrogen Deficiency ◽  
Culture Model

Platelet-Lysate for In Vitro Expansion of Human Multipotent Mesenchymal Stromal Cells in Approaches of Cell-Therapy.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2577-2577
Author(s):  
Maria Ester Bernardo ◽  
Maria Antonietta Avanzini ◽  
Cesare Perotti ◽  
Angela M. Cometa ◽  
Antonia Moretta ◽  
...  
Keyword(s):  
Immune Response ◽  
Stem Cell ◽  
Cell Therapy ◽  
Stromal Cells ◽  
Platelet Lysate ◽  
Proliferative Capacity ◽  
Regulatory Effect ◽  
Spontaneous Transformation ◽  
Hematopoietic Stem

Abstract There is large interest in the use of mesenchymal stromal cells (MSCs) in approaches of cell-therapy and tissue engineering. MSCs are currently expanded in vitro in the presence of fetal calf serum (FCS); however, FCS raises concerns in the case of clinical grade cellular preparations because of the theoretical risk of transmission of zoonoses and triggering immune reactions in the host. Therefore, the identification of a serum-free medium appropriate for both the extensive expansion necessary to reach the large numbers of MSCs required for clinical application, and the exclusion of risks connected with the use of animal products, is warranted. Aim of this study was to evaluate whether MSCs expanded in medium supplemented with platelet-lysate (PL) are endowed with biological properties appropriate for cell-therapy approaches. MSCs were generated from bone-marrow of 8 healthy hematopoietic stem cell donor; 4 different culture conditions were tested: 10 % FCS; 5% PL; 2,5% PL; 1% PL. MSCs were harvested when reaching ≥ 80% confluence and replated for expansion at 4.000 cells/cm² until passage 5. CFU-F frequency, proliferative capacity, morphology, surface phenotype and differentiation capacity were evaluated. In particular, the immune regulatory effect on alloantigen-specific immune response, the kinetics of cytokine production and the resistance to spontaneous transformation into tumor cells of MSC expanded in the presence of either PL or FCS were investigated. Our results demonstrate that MSCs expanded in either FCS or PL display comparable morphology, phenotype and differentiation capacity, while PL-MSCs were superior in terms of clonogenic efficiency and proliferative capacity. Immune-regulatory effect of MSCs was investigated on alloantigen-specific immune response in mixed lymphocyte culture (MLC). We found that MSCs-PL are comparable to MSCs-FCS in their capacity to: decrease alloantigen-induced cytotoxic activity; favor differentiation of CD4+ T-cell subsets expressing Treg phenotype; increase early secretion of IL-10 in MLC supernatant, as well as to induce a striking augmentation of IL-6 production. As compared with MSCs-PL, MSCs-FCS were more efficient in suppressing alloantigen-induced lymphocyte subset proliferation and in reducing early IFNg-secretion. Resistance to spontaneous transformation into tumor cells of expanded MSCs was demonstrated by both molecular karyotyping (array-comparative genomic hybridization) and maintenance of normal morphology/phenotype after prolonged in vitro culture. Our data support the hypothesis of a remarkable immune functional plasticity of human MSCs and suggest that the use of MSCs-PL, which seem to be endowed with a relatively low immune suppressive activity, could be more appropriate in approaches of reparative/regenerative cell-therapy or in strategies aimed at improving hematopoietic/immune recovery after hematopoietic stem cell transplantation (HSCT). On the contrary, as MSCs-FCS seem to display a more pronounced immune suppressive function, they might be more suitable for preventing or treating alloreactive-related immune complications, such as severe Graft-versus-host disease (GvHD) in HSCT and graft rejection in HSCT and solid organ transplantation.

A 3-Dimensional Co-Culture Model to Investigate Adhesion-Mediated Drug Resistance in Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1826-1826
Author(s):  
Omar S. Aljitawi ◽  
Yinghua Xiao ◽  
Karthik Ramachandran ◽  
Brea C. Lipe ◽  
Rama Garimella ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Stromal Cells ◽  
Culture Conditions ◽  
Lambda Light Chain ◽  
Ki 67 ◽  
Blocking Antibody ◽  
3 Dimensional ◽  
Culture Model ◽  
D Model

Abstract Abstract 1826 Background: In multiple myeloma (MM), evidence suggests that the adhesion of malignant plasma cells to bone marrow microenvironment elements is central to MM growth and drug resistance, otherwise referred to as adhesion–mediated drug resistance. As other investigators have shown, the MM-stroma interaction is in part mediated by adhesion molecules like N-cadherin. Goal: To develop a 3-dimensional MM in vitro co-culture model where MM cells interact with stromal elements mimicking the bone marrow microenvironment and use the model to investigate N-cadherin mediated drug resistance. Methods: We co-cultured U266 cells, a MM cell line, with mesenchymal stromal cells (MSCs) isolated from Wharton's jelly (WJ) in a synthetic copolymer (polyglycolic acid/ poly L-lactic acid 90/10 copolymer) (3-dimensional culture conditions). For controls, we cultured U266 cells over a monolayer of WJMCS (2-dimensional conditions) or U266 cells in suspension. Cell proliferation was measured by Ki-67 immunohistochemistry (IHC) stain and lambda light chain expression was measure by enzyme-linked immunosorbent assay (ELISA) and IHC stains. Bortezomib in 1, 5, and 10 nM concentrations was used to treat U266 cells for 24 hours under the three culture conditions. Cytotoxicity was measured by modified Alamar Blue assay. Results: U266 cells in our 3-D model adhered to stromal cells and formed clusters close to matrix material. U266 cells proliferated as evidenced by positive Ki-67 stain, and expressed lambda light chain by IHC and ELISA. Lambda light expression measured by ELISA peaked at 1 week of co-culture in the 3-D model. Though N-cadherin was expressed in both stromal cells and U266 MM cells in 3-D, it was expressed only in stromal cells in 2-D culture conditions. Finally, pre-incubating U266 cells with N-cadherin blocking antibody resulted in less population of the 3-D model by U266 MM cells. Treatment of U266 with bortezomib resulted in less cell cytotoxicity in 3-D and 2-D co-culture conditions compared to U266 cells treated in suspension. Conclusions: Using a 3-D in vitro co-culture model, we demonstrated that only 3-D co-culture conditions resulted in N-cadherin expression in U266 MM cell line. We also demonstrated that U266 population of the 3-D model was successfully blocked using an N-cadherin blocking antibody. We propose N-cadherin mediated adhesion as a mechanism to explain reduced cytotoxicity to bortezomib in 3-D. We propose this model to be used to investigate adhesion-mediated drug resistance. Disclosures: No relevant conflicts of interest to declare.

Establishment of a Primary Culture Model of Mouse Uterine and Vaginal Stroma for Studying In Vitro Estrogen Effects

2006 ◽  
Vol 231 (3) ◽  
pp. 303-310 ◽  
Author(s):  
Keiko Inada ◽  
Shinji Hayashi ◽  
Taisen Iguchi ◽  
Tomomi Sato
Keyword(s):  
Cell Proliferation ◽  
Epithelial Cell ◽  
Primary Culture ◽  
Stromal Cells ◽  
Renal Capsule ◽  
Epithelial Cell Proliferation ◽  
Culture Model ◽  
Brdu Labeling

Effects of 17β-estradiol (E2) on uterine and vaginal epithelial cell proliferation could be mediated by stromal cell-derived paracrine factors. To study the epithelial-stromal interactions in mice, an in vitro model of uterine and vaginal stromal cells of immature mice is essential. Therefore, we established a primary culture model of stromal cells both from uterus and vagina and examined the effect of E2 on proliferation of cultured stromal cells. We found that E2 stimulated proliferation of stromal cells from both organs in vitro, showing an increase in the number of cells and the percentage of 5-bromo-2'-deoxyurldine (BrdU)-labeled cells. Interestingly, vaginal stromal cells responded to lower E2 than uterine stromal cells in proliferation (10−12 M vs. 10−8 M) and BrdU labeling (10−14 -10−10 M vs. 10−10 - 10−6 M). To examine the effect of E2 in vivo, cells were grafted into the subrenal capsule of the host mice and grown for 2 weeks. The BrdU labeling in cultured stromal cells was increased by E2 in vivo. To examine the effect of cultured stromal cells on epithelial cell proliferation, uterine and vaginal epithelium of adult mice were separated, recombined with the cultured stromal cells, and grafted under the renal capsule of hosts for 3 weeks. Epithelial cells recombined with cultured stromal cells showed simple columnar morphology in uterine grafts and stratified and keratinized morphology in vaginal grafts under the influence of the hormonal environment of the hosts. The BrdU labeling in epithelial cells was increased by E2, suggesting that cultured stromal cells can stimulate epithelial cell proliferation. In conclusion, we established a primary culture model of uterine and vaginal stromal cells, which can be mitogenically stimulated by E2 in vitro and in vivo after being grafted under the renal capsule. This culture system will be useful for investigating the underlying molecular mechanisms of uterine and vaginal epithelial-stromal interactions.

Sign in / Sign up

Export Citation Format

Share Document