182 In Vitro Derivation of Male Germ Cells from Bovine Bone Marrow-Derived Mesenchymal Stem Cells

2018 ◽  
Vol 30 (1) ◽  
pp. 231
Author(s):  
J. Cortez ◽  
J. Bahamonde ◽  
J. Palomino ◽  
M. De los Reyes ◽  
C. Torres ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Germ Cell ◽  
Mrna Expression ◽  
Male Germ Cell ◽  
Bovine Bone ◽  
Mrna Levels ◽  
Germ Cell Differentiation

During the last few years, the in vitro derivation of germ cell lineages from stem cells has emerged as an exciting new strategy for obtaining mature gametes. In vitro gamete derivation technology has potential applications as an alternative method for dissemination of elite animal genetics, production of transgenic animals, and conservation of endangered species. Germ cell differentiation and gametogenesis is a complex process and potential of different stem cell donors (i.e. SSC, ESC, iPSC) for in vitro male germ cell derivation has been inconsistent. Mesenchymal stem cells (MSC) may be suitable candidates for in vitro gamete derivation considering their (1) plasticity that is not limited to mesodermal derivatives, (2) availability of abundant tissues sources for isolation, (3) high proliferative potential, (4) simple and inexpensive isolation, and (5) high potential for cell therapy, including autologous or allogenic transplantation. The present study aimed to induce differentiation of MSC isolated from bone marrow derived from bovine male fetuses (bfMSC) into the germ cell lineage using an in vitro approach based on the exogenous effect of retinoic acid (RA) and bone morphogenetic protein 4 (BMP4). Differentiation media consisted in control media (DMEM with high glucose plus 10% fetal bovine serum, 100 IU mL−1 penicillin, 100 μg mL−1 streptomycin, and 0.25 μg mL−1amphotericin B) supplemented with RA (0.01, 0.1, or 1 µM) or BMP4 (10, 50, or 100 ng mL−1). Cell samples were obtained from differentiating and control bfMSC cultures and analysed for expression of housekeeping genes β-ACTIN and GAPDH, pluripotent genes OCT4 and NANOG, germ cell genes FRAGILLIS, STELLA, and VASA, male germ cell genes DAZL, PIWIl2, and STRA8, and meiotic biomarker SCP3 by quantitative-PCR (Q-PCR). OCT4, NANOG, and DAZL were immunodetected in undifferentiated and differentiated bfMSC using flow-cytometry analysis. The mRNA expression of DAZL was activated by RA or BMP4 supplementation, although no differences (P > 0.05) were detected among different concentrations. DAZL and NANOG mRNA levels increased (P < 0.05) from Day 7 to Day 21 during supplementation of RA (0.1 μM). In comparison, DAZL mRNA levels increased (P < 0.05) at Day 14 during supplementation of BMP4 (100 ng). OCT4 and SCP3 mRNA levels were not affected by RA or BMP4 treatments. Transcripts of FRAGILLIS, STELLA, VASA, PIWIl2, and STRA8 were not detected in control or differentiated bfMSC. Higher (P < 0.05) percentages of undifferentiated bfMSC were positive for NANOG (80.6%) and OCT4 (83.4%). DAZL- and NANOG-positive cells were 2.1% and 2.9%, and 95.9% and 97.8% at Days 0 and 21 of RA treatment, respectively. Data indicated that expression of germ cell biomarker DAZL in bfMSC is activated and increased after in vitro supplementation of RA and BMP4. Moreover, NANOG mRNA levels were regulated by RA treatment. Similar levels of SCP3 mRNA expression suggest that differentiated bfMSC were not induced into meiosis. Thus, exposure of bfMSC to RA or BMP4 under in vitro conditions might induce an early stage of premeiotic germinal differentiation.

217 PROTEIN-INDUCED TRANSDIFFERENTIATION INTO MALE GERM CELL-LIKE LINEAGE FROM CHICKEN FETAL BONE MARROW STEM CELLS

2012 ◽  
Vol 24 (1) ◽  
pp. 220
Author(s):  
J. M. Yoo ◽  
J. J. Park ◽  
K. Gobianand ◽  
J. Y. Ji ◽  
J. S. Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Retinoic Acid ◽  
Germ Cell ◽  
Germ Cells ◽  
Cultured Cells ◽  
Male Germ Cell ◽  
Male Germ Cells ◽  
Germ Cell Differentiation ◽  
Transgenic Chickens

Bone marrow (BM)-derived stem cells are capable of transdifferentiation into multilineage cells like muscle, bone, cartilage, fat and nerve cells. In this study, we investigated the capability of mesenchymal stem cells (MSC) derived from BM into germ cell differentiation in the chicken. Chicken MSCs were isolated from BM of day 20 fertilized fetal chicken with Ficoll-Paque Plus. Isolated cells were cultured in advance-DMEM (ADMEM) supplemented with 10% fetal bovine serum and antibiotics. Once confluent, cells were subcultured until five passages. The cultured cells showed fibroblast-like morphology. The cells had positive expressions of Oct4, Sox2 and Nanog. Two induction methods were conducted to examine the ability of transdifferentation into male germ cells. In group 1, MSC were cultured in ADMEM containing retinoic acid and chicken testicular extracts proteins for 10 to 15 days. In group 2, MSC were permeabilized by streptolysin O and treated with chicken testicular protein extracts. In both treatment groups, MSC were cultured in ADMEM containing retinoic acid for 10 to 15 days. We found that chicken MSC had a positive expression of pluripotent proteins such as Oct4, Sox2, Nanog and a small population of chicken MSC seem to transdifferentiate into male germ cell-like cells. These cells expressed early germ cell markers and male germ-cell-specific markers (Dazl, C-kit, Stra8 and DDX4) as analysed by reverse transcription-PCR and immunohistochemistry. These results demonstrated that chicken MSC may differentiate into male germ cells and the same might be used as a potential source of cells for production of transgenic chickens. This study was carried out with the support of Agenda Program (Project No. PJ0064692011), RDA and Republic of Korea.

scholarly journals Curcumin Alleviates the Senescence of Canine Bone Marrow Mesenchymal Stem Cells during In Vitro Expansion by Activating the Autophagy Pathway

2021 ◽  
Vol 22 (21) ◽  
pp. 11356
Author(s):  
Jiaqiang Deng ◽  
Ping Ouyang ◽  
Weiyao Li ◽  
Lijun Zhong ◽  
Congwei Gu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Mrna Expression ◽  
Light Chain ◽  
Therapeutic Potential ◽  
Cell Model ◽  
Biological Aging ◽  
Aging Effects

Senescence in mesenchymal stem cells (MSCs) not only hinders the application of MSCs in regenerative medicine but is also closely correlated with biological aging and the development of degenerative diseases. In this study, we investigated the anti-aging effects of curcumin (Cur) on canine bone marrow-derived MSCs (cBMSCs), and further elucidated the potential mechanism of action based on the modulation of autophagy. cBMSCs were expanded in vitro with standard procedures to construct a cell model of premature senescence. Our evidence indicates that compared with the third passage of cBMSCs, many typical senescence-associated phenotypes were observed in the sixth passage of cBMSCs. Cur treatment can improve cBMSC survival and retard cBMSC senescence according to observations that Cur (1 μM) treatment can improve the colony-forming unit-fibroblasts (CFU-Fs) efficiency and upregulated the mRNA expression of pluripotent transcription factors (SOX-2 and Nanog), as well as inhibiting the senescence-associated beta-galactosidase (SA-β-gal) activities and mRNA expression of the senescence-related markers (p16 and p21) and pro-inflammatory molecules (tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)). Furthermore, Cur (0.1 μM~10 μM) was observed to increase autophagic activity, as identified by upregulation of microtubule-associated protein 1 light chain 3 (LC3), unc51-like autophagy-activating kinase-1 (ULK1), autophagy-related gene (Atg) 7 and Atg12, and the generation of type II of light chain 3 (LC3-II), thereby increasing autophagic vacuoles and acidic vesicular organelles, as well as causing a significant decrease in the p62 protein level. Moreover, the autophagy activator rapamycin (RAP) and Cur were found to partially ameliorate the senescent features of cBMSCs, while the autophagy inhibitor 3-methyladenine (3-MA) was shown to aggravate cBMSCs senescence and Cur treatment was able to restore the suppressed autophagy and counteract 3-MA-induced cBMSC senescence. Hence, our study highlights the important role of Cur-induced autophagy and its effects for ameliorating cBMSC senescence and provides new insight for delaying senescence and improving the therapeutic potential of MSCs.

scholarly journals Cellular Proliferation of Equine Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stem Cells Decline With Increasing Donor Age

2020 ◽  
Vol 7 ◽  
Author(s):  
Jasmin Bagge ◽  
James N. MacLeod ◽  
Lise C. Berg
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Cellular Proliferation ◽  
Age Groups ◽  
Mrna Levels ◽  
Donor Age ◽  
Autologous Cell

Background: Bone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stem cells (MSCs) are used increasingly for autologous cell therapy in equine practice to treat musculoskeletal and other injuries. Current recommendations often call for 10–100 million MSCs per treatment, necessitating the expansion of primary cells in culture prior to therapeutic use. Of concern, human and rodent studies have shown a decline of both MSC recovery from sampled tissue and in vitro proliferative capacity with increasing donor age. This may be problematic for applications of autologous cell-based therapies in the important equine demographic of older patients.Objectives: To investigate the effect of donor age on the cellular proliferation of equine BM- and AT-MSCs.Study Design:In vitro study.Methods: BM- and AT-MSCs and dermal fibroblasts (biological control) were harvested from horses in five different age groups (n = 4, N = 60); newborn (0 days), yearling (15–17 months), adult (5–8 years), middle-aged (12–18 years), and geriatric (≥22 years). Proliferation of the cells was tested using an EdU incorporation assay and steady state mRNA levels measured for targeted proliferation, aging, and senescence biomarkers.Results: The cellular proliferation of equine BM- and AT-MSCs declined significantly in the geriatric cohort relative to the younger age groups. Proliferation levels in the two MSC types were equally affected by donor age. Analysis of steady state mRNA levels showed an up-regulation in tumor suppressors, apoptotic genes, and multiple growth factors in MSCs from old horses, and a down-regulation of some pro-cycling genes with a few differences between cell types.Main Limitations: Potential age-dependent differences in cell function parameters relevant to cell-therapy application were not investigated.Conclusions: The cellular proliferation of equine BM- and AT-MSCs declined at advanced donor ages. High levels of in vitro proliferation were observed in both MSC types from horses in the age groups below 18 years of age.

Germ cell differentiation of bone marrow mesenchymal stem cells

Andrologia ◽  
2019 ◽  
Vol 51 (4) ◽  
pp. e13229 ◽  
Author(s):  
Maryam Salem ◽  
Tooba Mirzapour ◽  
Abolfazl Bayrami ◽  
Mohsen Sagha
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Cell Differentiation ◽  
Germ Cell ◽  
Germ Cell Differentiation ◽  
Marrow Mesenchymal Stem Cells

scholarly journals Rat bone marrow derived mesenchymal stem cells differentiate to germ cell like cells

2018 ◽  
Author(s):  
Kuldeep Kumar ◽  
Kinsuk Das ◽  
AP Madhusoodan ◽  
Ajay Kumar ◽  
Purnima Singh ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Animal Species ◽  
Germ Plasm ◽  
Domestic Animal ◽  
Rat Bone

Summary StatementThe in vitro differentiation of rMSCs provides an excellent model for studying cell commitment and their potential in stem cell technology. We have demonstrated that rat bone marrow derived MSCs are able to differentiate into germ-line cells in vitro which has an enormous scope in the advancement of fertility research. In future, this technique can be utilized in different domestic animal species for propagation of elite germ plasm.AbstractGerm cells undergo distinctive male or female pathways to produce spermatozoa or oocyte respectively essential for sexual reproduction. Mesenchymal stem cells (MSCs) have the capacity of trans-differentiation to form the multiple lineages of cells of mesoderm, endoderm, and ectoderm origin. Herein, MSCs were isolated from rat bone marrow and characterized by their morphological features, expression of surface markers by immunophenotyping and molecular biology tools as well as self renewal and differentiation capability. Thereafter, by inducing these cells with retinoic acid we could able to show that bone marrow derived MSCs are able to trans-differentiate into male germ cell-like cells which were further characterized by the expressions of germ cell specific markers. This in vitro study for the generation of germ-like cells suggests that bone marrow MSCs can be a potential source of germ cells that could be a sustainable source of sperm / oocyte production for potential therapeutic applications in future. Moreover, this technique can be applied in different domestic animal species for propagation of elite germ plasm.

The Supportive Role Of Mesenchymal Stem Cells On Normal Hematopoietic Stem Cells Is Impaired In Leukemic Bone Marrow

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1214-1214
Author(s):  
Meiguang Lin ◽  
Yakun Pang ◽  
Yawei Zheng ◽  
Xin Gu ◽  
Feng-chun Yang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Mrna Expression ◽  
Hematopoietic Stem ◽  
Supporting Function ◽  
Significant Difference ◽  
Leukemia Development

Abstract Mesenchymal stem cells (MSCs) have been shown to be able to form a niche that maintains hematopoietic stem cells (HSCs). However, how the phonotypical characteristics and biological functions of MSCs are affected and especially whether their supporting role on normal HSC is impaired in leukemic microenvironment, are not well defined. Here, we have examined the changes of biological characteristics of mouse MSCs and in particular their supporting function on normal HSCs in Notch1-induced acute T cell lymphocytic leukemia (T-ALL) mice (Hu et al, Blood 2009; 114:3783-3792). MSCs were sorted based on the phenotypic markers, PDGFRα+Sca-1+CD45-TER119- (PαS) (Houlihan et al, Nat Protoc 2012) from control or T-ALL mice and flow cytometric analysis was conducted at different time points during leukemia development. Our results showed there was no significant difference between T-ALL and control mice regarding the absolute number and percentage of PαS MSCs in total bone marrow mono-nuclear cells (BMMNCs), cell cycling status and the percentage of apoptosis of freshly sorted PαS MSCs. Moreover, PαS MSCs from both normal and T-ALL leukemic marrow had similar morphology (spindle and polymorphic shaped), and uniformly expressed known cell surface markers for cultured mouse MSCs (CD29, CD44, Sca-1). However, the number of T-ALL PαS MSCs colony forming unit-fibroblastic (CFU-F) formation was 3-fold lower than control CFU-F (p<0.001). Levels of mRNA expression of genes associated with adipogenic (adipsin, mLP, PPARγ), osteogenic (Bmp4, Sp7, Bglap) and chondrogenic (collagen II, collagen X, aggrecan) differentiation in PαS MSCs were significantly decreased in T-ALL mice compared with control mice (p<0.05). These results suggest that the proliferation potential and the differentiation potential of MSC were decreased in the leukemic environment. To investigate whether the decrease in the proliferation of T-ALL PαS MSCs was associated with a change in cellular senescence, β-galactosidase activity and quantitative RT-PCR analysis of genes associated with senescence were performed. Our finding showed a significant increase in the number of β-galactosidase–positive cells (control/T-ALL; 3.11±0.20%/3.99±0.08%, p<0.05) and in the mRNA expression level of senescence-related gene p16 in PαS MSCs T-ALL mice compared with control mice (control/T-ALL; 1.00±0.03/1.57±0.17, p<0.05). These results imply that p16 plays an important role in PαS MSCs senescence in T-ALL microenvironment. To determine whether the supporting function of T-ALL PαS MSCs on normal HSCs proliferation is impaired, the cobble-stone area forming cell (CAFC) assay, an in vitro surrogate for HSCs, was applied. Normal control or T-ALL PαS MSCs were co-cultured with the same pool of normal HSCs. After 5 weeks, co-cultures containing normal PαS MSC formed multiple large CAFCs. On the contrast, T-ALL PαS MSCs supported 7-fold less hypocellular CAFCs (p<0.05). This data indicate that T-ALL PαS MSCs have a reduced ability to support normal HSCs proliferation in vitro. To further validate the change of PαS MSCs’ supporting function on normal HSCs proliferation in leukemic microenvironment observed in vitro, in vivo co-transplantation study were performed. Our preliminary results further indicated that the supporting function of MSCs on normal hematopoiesis in the leukemic microenvironment was compromised. In conclusion, dysfunction of MSC, an important component of HSC microenvironment, may play a crucial role in the suppression of HSC during leukemia development. Improving the function of MSC may serve as a new strategy to enhance normal hematopoiesis in leukemic marrow. Disclosures: No relevant conflicts of interest to declare.

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