scholarly journals Anabolic Steroids-Driven Regulation of Porcine Ovarian Putative Stem Cells Favors the Onset of Their Neoplastic Transformation

2021 ◽  
Vol 22 (21) ◽  
pp. 11800
Author(s):  
Gabriela Gorczyca ◽  
Kamil Wartalski ◽  
Jerzy Wiater ◽  
Marcin Samiec ◽  
Zbigniew Tabarowski ◽  
...  
Keyword(s):  
Stem Cells ◽  
Nuclear Transfer ◽  
Ex Vivo ◽  
Anabolic Steroids ◽  
Neoplastic Transformation ◽  
Donor Cells ◽  
First Time ◽  
Different Doses ◽  
Cloned Pig

Nandrolone (Ndn) and boldenone (Bdn), the synthetic testosterone analogues with strong anabolic effects, despite being recognized as potentially carcinogenic compounds, are commonly abused by athletes and bodybuilders, which includes women, worldwide. This study tested the hypothesis that different doses of Ndn and Bdn can initiate neoplastic transformation of porcine ovarian putative stem cells (poPSCs). Immunomagnetically isolated poPSCs were expanded ex vivo in the presence of Ndn or Bdn, for 7 and 14 days. Results show that pharmacological doses of both Ndn and Bdn, already after 7 days of poPSCs culture, caused a significant increase of selected, stemness-related markers of cancer cells: CD44 and CD133. Notably, Ndn also negatively affected poPSCs growth not only by suppressing their proliferation and mitochondrial respiration but also by inducing apoptosis. This observation shows, for the first time, that chronic exposure to Ndn or Bdn represents a precondition that might enhance risk of poPSCs neoplastic transformation. These studies carried out to accomplish detailed molecular characterization of the ex vivo expanded poPSCs and their potentially cancerous derivatives (PCDs) might be helpful to determine their suitability as nuclear donor cells (NDCs) for further investigations focused on cloning by somatic cell nuclear transfer (SCNT). Such investigations might also be indispensable to estimate the capabilities of nuclear genomes inherited from poPSCs and their PCDs to be epigenetically reprogrammed (dedifferentiated) in cloned pig embryos generated by SCNT. This might open up new possibilities for biomedical research aimed at more comprehensively recognizing genetic and epigenetic mechanisms underlying not only tumorigenesis but also reversal/retardation of pro-tumorigenic intracellular events.

scholarly journals Microbial Transglutaminase Improves ex vivo Adhesion of Gelatin Methacryloyl Hydrogels to Human Cartilage

2021 ◽  
Vol 3 ◽  
Author(s):  
Anna Trengove ◽  
Serena Duchi ◽  
Carmine Onofrillo ◽  
Cathal D. O'Connell ◽  
Claudia Di Bella ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ex Vivo ◽  
Surgical Techniques ◽  
Microbial Transglutaminase ◽  
Surrounding Tissue ◽  
Cartilage Defects ◽  
Human Cartilage ◽  
First Time

Current surgical techniques to treat articular cartilage defects fail to produce a satisfactory long-term repair of the tissue. Regenerative approaches show promise in their ability to generate hyaline cartilage using biomaterials in combination with stem cells. However, the difficulty of seamlessly integrating the newly generated cartilage with the surrounding tissue remains a likely cause of long-term failure. To begin to address this integration issue, our strategy exploits a biological enzyme (microbial transglutaminase) to effect bioadhesion of a gelatin methacryloyl implant to host tissue. Mechanical characterization of the bioadhesive material shows that enzymatic crosslinking is compatible with photocrosslinking, allowing for a dual-crosslinked system with improved mechanical properties, and a slower degradation rate. Biocompatibility is illustrated with a 3D study of the metabolic activity of encapsulated human adipose derived stem cells. Furthermore, enzymatic crosslinking induced by transglutaminase is not prevented by the presence of cells, as measured by the bulk modulus of the material. Adhesion to human cartilage is demonstrated ex vivo with a significant increase in adhesive strength (5.82 ± 1.4 kPa as compared to 2.87 ± 0.9 kPa, p < 0.01) due to the addition of transglutaminase. For the first time, we have characterized a bioadhesive material composed of microbial transglutaminase and GelMA that can encapsulate cells, be photo crosslinked, and bond to host cartilage, taking a step toward the integration of regenerative implants.

scholarly journals Dynamic integration of enteric neural stem cells in ex vivo organotypic colon cultures

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Georgina Navoly ◽  
Conor J. McCann
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Organotypic Culture ◽  
Ex Vivo ◽  
Donor Cell ◽  
Colonic Tissue ◽  
Cell Therapies ◽  
Enteric Ganglia ◽  
Donor Cells

AbstractEnteric neural stem cells (ENSC) have been identified as a possible treatment for enteric neuropathies. After in vivo transplantation, ENSC and their derivatives have been shown to engraft within colonic tissue, migrate and populate endogenous ganglia, and functionally integrate with the enteric nervous system. However, the mechanisms underlying the integration of donor ENSC, in recipient tissues, remain unclear. Therefore, we aimed to examine ENSC integration using an adapted ex vivo organotypic culture system. Donor ENSC were obtained from Wnt1cre/+;R26RYFP/YFP mice allowing specific labelling, selection and fate-mapping of cells. YFP+ neurospheres were transplanted to C57BL6/J (6–8-week-old) colonic tissue and maintained in organotypic culture for up to 21 days. We analysed and quantified donor cell integration within recipient tissues at 7, 14 and 21 days, along with assessing the structural and molecular consequences of ENSC integration. We found that organotypically cultured tissues were well preserved up to 21-days in ex vivo culture, which allowed for assessment of donor cell integration after transplantation. Donor ENSC-derived cells integrated across the colonic wall in a dynamic fashion, across a three-week period. Following transplantation, donor cells displayed two integrative patterns; longitudinal migration and medial invasion which allowed donor cells to populate colonic tissue. Moreover, significant remodelling of the intestinal ECM and musculature occurred upon transplantation, to facilitate donor cell integration within endogenous enteric ganglia. These results provide critical evidence on the timescale and mechanisms, which regulate donor ENSC integration, within recipient gut tissue, which are important considerations in the future clinical translation of stem cell therapies for enteric disease.

257 EXPRESSION OF DEVELOPMENTAL GENES IN PORCINE NUCLEAR TRANSFER EMBRYOS RECONSTRUCTED WITH BONE MARROW MESENCHYMAL STEM CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 236
Author(s):  
B. Mohana Kumar ◽  
H.-F. Jin ◽  
J.-G. Kim ◽  
S. Balasubramanian ◽  
S.-Y. Choe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Nuclear Transfer ◽  
Expression Profiles ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Marrow Mesenchymal Stem Cells

Abnormal gene expression is frequently observed in nuclear transfer (NT) embryos and is one of the suggested causes of the low success rates of this approach. Recent study has suggested that adult stem cells may be better donor cells for NT, as their less differentiated state may ease epigenetic reprogramming by the oocyte (Kato et al. 2004 Biol. Reprod. 70, 415-418). In the present study, we investigated the expression profile of some selected genes involved in the development of the pre-implantation embryos of in vivo- and NT-derived origin using bone marrow mesenchymal stem cells (MSCs) and porcine fetal fibroblasts (pFF) as donors. Isolated population of MSCs from porcine bone marrow were characterized by cell-surface antigen profile (CD13pos, CD105pos, CD45neg, and CD133neg) and by their extensive consistent differentiation to multiple mesenchymal lineages (adipocytic, osteocytic and chondrocytic) under controlled in vitro conditions (Pittenger et al. 1999 Science 284, 143-147). Primary cultures of pFF from a female fetus at <30 days of gestation were established. for NT, donor cells at 3-4 passages were employed. Embryos cloned from MSCs showed enhanced developmental potential compared to pFF cloned embryos, indicated by higher rates of blastocyst formation (15.3% � 4.8 and 9.0% � 3.9, respectively) and total cell number (31.5 � 7.2 and 20.5 � 5.4, respectively) in Day 7 blastocysts. Total RNA was extracted from pools (triplicates) of 10 embryos each of 8-cell, morula, and blastocyst stages of in vivo and NT origin using Dynabeads� mRNA DIRECT" kit (Dynal, Oslo, Norway). Reverse transcription was performed with a Superscript" III cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA). Real-time PCR was performed on a Light cycler� using FastStart DNA Master SYBR Green I (Roche Diagnostics, Mannheim, Germany). The expression profiles of genes involved in transcription (Oct-4, Stat3), DNA methylation (Dnmt1), de novo methylation (Dnmt3a), histone deacetylation (Hdac2), anti-apoptosis (Bcl-xL), and embryonic growth (Igf2r) were determined. The mRNA of H2a was employed to normalize the levels. Significant differences (P < 0.05) in the relative abundance of Stat3, Dnmt1, Dnmt3a, Bcl2, and Igf2r were observed in pFF NT embryos compared with in vivo-produced embryos, whereas embryos derived from MSCs showed expression patterns similar to those of in vivo-produced embryos. However, Oct-4 and Hdac2 revealed similar expression profiles in NT- and in vivo-produced embryos. These results indicate that MSC-derived NT embryos had enhanced embryonic development and their gene expression pattern more closely resembled that of in vivo-produced embryos. Hence, less differentiated MSCs may have a more flexible potential in improving the efficiency of the porcine NT technique. This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.

VHL-mutant renal cell carcinomas contain cancer cells with mesenchymal phenotypes.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 4568-4568 ◽  
Author(s):  
Craig Gedye ◽  
Danylo Sirskyj ◽  
Nazleen Carol Lobo ◽  
Andrew Evans ◽  
Neil Eric Fleshner ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cancer Stem Cells ◽  
Cancer Cells ◽  
Tumor Cells ◽  
Renal Cell ◽  
Clear Cell ◽  
Ex Vivo ◽  
Functional Characterization ◽  
Mutant Cells

4568 Background: To study “cancer stem cells” it is imperative to account for all stromal cell populations within the tumour. The existence of “cancer stem cells” in clear cell renal cell carcinoma (ccRCC) has not been examined in ex vivo patient samples. Methods: We established a multiplex flow cytometry (FC) antibody panel in ccRCC, which reliably identified stromal lineages including CD45+ immune, CD31+/CD144+ endothelial and fibroblast-marker-positive subpopulations, thus allowing isolation of "lineage-negative" tumor cells. To verify the identity of tumour-derived populations as either cancer cells or normal stromal cells, we took advantage of the fact that mutations in VHL occur early during ccRCC tumorigenesis and are found in two-thirds of patients. Results: We sequenced 18 patient tumor samples, 12 of which had VHL exome mutations. Targeted re-sequencing of FC sorted subpopulations from these patients’ samples revealed that while CD45+ immune cells and CD31+/CD144+ endothelial cells were genetically normal, a population of VHL-mutant fibroblast-marker positive cells was consistently identified in every patient’s tumour. Immunohistochemistry showed that fibroblast marker-positive VHL-mutant cells do not have the large “clear cell” morphology typical of the majority of the cancer cells in these tumours. When purified and cultured, these fibroblast marker-positive VHL-mutant cells proliferate extensively under mesenchymal culture conditions, but displayed different morphologies to lineage-negative VHL-mutant tumor cells. Functional characterization of these FC sorted cell subpopulations is ongoing, including proliferation, migration, invasion, differentiation and treatment resistance. Conclusions: The phenotype and preliminary functional characterization of these VHL-mutant fibroblast-marker positive cells suggests a mesenchymal differentiation program in ccRCC, with implications for the ontogeny, biology and clinical management of VHL-mutant renal cancer.

Donor-derived DNA in fingernails among recipients of allogeneic hematopoietic stem-cell transplants

Blood ◽  
2007 ◽  
Vol 110 (7) ◽  
pp. 2231-2234 ◽  
Author(s):  
Daisuke Imanishi ◽  
Yasushi Miyazaki ◽  
Reishi Yamasaki ◽  
Yasushi Sawayama ◽  
Jun Taguchi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Blood Cells ◽  
Hematopoietic Stem ◽  
Donor Cells ◽  
Cell Transplants ◽  
First Time ◽  
Hematopoietic Stem Cell Transplants ◽  
Short Tandem

To examine whether donor-derived cells could exist in nonhematopoietic tissues of recipients after allogeneic hematopoietic stem-cell transplantation, we examined the patterns of the short tandem repeat (STR) of DNA extracted from fingernail clippings of recipients so that the contamination of blood cells was excluded. All 21 patients reached donor-derived hematopoiesis after transplantation and 20 of them were in remission of the primary diseases at the time of sampling. Compared with the STRs of donor cells, among 9 of 21 patients, DNA extracted from fingernail samples showed coexistence of the donor pattern of the STRs, sharing from 8.9% to 72.9% of total STR areas. Time from transplantation to sampling was from 305 to 2399 days among positive cases. These results demonstrate for the first time the existence of stable contribution of donor cells in fingernails among recipients of allogeneic hematopoietic stem cells.

scholarly journals Impact of source tissue and ex vivo expansion on the characterization of goat mesenchymal stem cells

2015 ◽  
Vol 6 (1) ◽  
pp. 1 ◽  
Author(s):  
Nuradilla Mohamad-Fauzi ◽  
Pablo J Ross ◽  
Elizabeth A Maga ◽  
James D Murray
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Source Tissue

scholarly journals Characterization of Ex Vivo Expanded Oral Mucosal Epithelium Cells on Acellular Porcine Corneal Stroma for Ocular Surface Reconstruction

2017 ◽  
Vol 2017 ◽  
pp. 1-8 ◽  
Author(s):  
Jia-Song Wang ◽  
Hua-Tao Xie ◽  
Ming-Chang Zhang
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Ex Vivo ◽  
Corneal Stroma ◽  
Feeder Cells ◽  
Rt Pcr ◽  
Phenotypic Characteristics ◽  
Mucosal Epithelium ◽  
Epithelium Cells

Purpose. To ex vivo expand oral mucosal epithelium cells (OMECs) on acellular porcine corneal stroma (APCS) without using feeder cells and serum and to compare the morphologic and phenotypic characteristics of cultured oral cells on APCS to those of cells on deluded human amniotic membrane (HAM). Methods. SD rat oral mucosal biopsies were cultured on APCS and HAM. Reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry were used to analyze the characterization of stem cells and epithelial differentiation of the outgrowth products. Results. Stratified and optimal transplantable OMECs were obtained after being cultured three to four weeks. Both RT-PCR and immunohistochemistry showed that cultured OMECs expressed markers of epithelial differentiation cytokeratin K3 and epithelial stem cell markers of p63 and ABCG2. Conclusions. OMECs can be successfully cultured on APCS without using xenobiotic feeder cells and serum. Characterization showed that these sheets retain the morphologic and phenotypic characteristics of OMECs within differentiated cells and stem cells. The optimal transplantable sheets can prove to be particularly beneficial to both bilateral limbal stem cell deficiency and deep corneal lesions.

Characterization of Purified and Ex Vivo Manipulated Human Hematopoietic Progenitor and Stem Cells in Xenograft Recipients

1999 ◽  
Vol 872 (1 HEMATOPOIETIC) ◽  
pp. 200-210 ◽  
Author(s):  
THOMAS A. BOCK ◽  
BENEDIKT L. ZIEGLER ◽  
HANS-JORG BUHRING ◽  
STEFAN SCHEDING ◽  
WOLFRAM BRUGGER ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ex Vivo ◽  
Hematopoietic Progenitor

scholarly journals Characterization of the secretory profile and exosomes of limbal stem cells in the canine species

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0244327
Author(s):  
Antonio J. Villatoro ◽  
Cristina Alcoholado ◽  
María del Carmen Martín-Astorga ◽  
Gustavo Rico ◽  
Viviana Fernández ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ocular Surface ◽  
Inhibitory Effect ◽  
Proteomic Profile ◽  
Limbal Stem Cells ◽  
High Concentrations ◽  
First Time

Limbal stem cells (LSCs) are a quiescent cell population responsible for the renewal of the corneal epithelium. Their deficiency is responsible for the conjunctivization of the cornea that is seen in different ocular pathologies, both in humans and in the canine species. The canine species represents an interesting preclinical animal model in ocular surface pathologies. However, the role of LSCs in physiological and pathological conditions in canine species is not well understood. Our objective was to characterize for the first time the soluble factors and the proteomic profile of the secretome and exosomes of canine LSCs (cLSCs). In addition, given the important role that fibroblasts play in the repair of the ocular surface, we evaluated the influence of the secretome and exosomes of cLSCs on their proliferation in vitro. Our results demonstrated a secretory profile of cLSCs with high concentrations of MCP-1, IL-8, VEGF-A, and IL-10, as well as significant production of exosomes. Regarding the proteomic profile, 646 total proteins in the secretome and 356 in exosomes were involved in different biological processes. Functionally, the cLSC secretome showed an inhibitory effect on the proliferation of fibroblasts in vitro, which the exosomes did not. These results open the door to new studies on the possible use of the cLSC secretome or some of its components to treat certain pathologies of the ocular surface in canine species.

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