scholarly journals Female Bioengineering: Primordial Germ Cell Differentiation of Mesenchymal Stem Cells onto Placental Scaffolds

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Amanda Baracho Trindade Hill ◽  
Antonio Alexandre Speri Alves ◽  
Rodrigo da Silva Nunes Barreto ◽  
Fabiana Fernandes Bressan ◽  
Maria Angelica Miglino ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Low Cost ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Isolated Cells ◽  
Umbilical Vessel ◽  
Germ Cell Differentiation ◽  
Cell Scaffold ◽  
Wide Availability

Introduction: Female infertility is a condition that is currently treated through the maximization of existing reserves; a necessity due to the fact that a true reversal of the processes leading to infertility is not yet technologically possible. This experiment examined the ability of mesenchymal stem cells to differentiate into primordial germ cells (PGC) when cultured onto the placental scaffold. Methods: To produce the scaffolds, the cotelydons were collected and decellularized by umbilical vessel SDS perfusion. Adipose derived cells were isolated based on rapid adherence to the plastic. Results: The isolated cells displayed the markers CD90 and 105, while lacked CD34 and CD45. When seeded onto the scaffold, the cells successfully differentiated into PGC like cells, displaying typical PGCs markers, including STELLA, OCT4, DAZL and VASA. Discussion: These materials were chosen for their low cost and wide availability. The data in this study show the promising potential of cell-scaffold complex to support the development of female tissue engineering-based regenerative medicine therapies.

scholarly journals Characterization of neuronal precursors obtained from human adipose-derived mesenchymal stem cells

2021 ◽  
Vol 31 (Supplement_2) ◽  
Author(s):  
N B Oliveira ◽  
A C Irioda ◽  
P E F Stricker ◽  
B F Mogharbel ◽  
N N Rosa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Neural Cells ◽  
High Plasticity ◽  
Rt Pcr ◽  
Isolated Cells ◽  
Neural Precursors ◽  
Hla Dr ◽  
Neuronal Precursors ◽  
Neural Growth

Abstract Background Mesenchymal stem cells (MSCs) can be isolated from any tissue derived from the mesoderm and have as main characteristics: high plasticity, the ability to originate mesodermal and non-mesodermal tissues, acting in the modulation of the inflammatory response, and the tissue repair. When grown in microenvironments with elasticity comparable to the human brain, these cells can differentiate efficiently in neural cells due to the mechanism related to the YAP protein, which can mediate responses to substrate stiffness in mesenchymal stem cells. Methods Human adipose-derived MSCs were isolated*, then it was done the trilineage test into adipocytes, osteocytes and, chondrocytes. Besides that, differentiation to neural precursor cells was through neurospheres after seeding the cells over a natural biopolymer matrix as NFBX. Those cells were analyzed using flow cytometry for the surface markers CD13, CD34, CD45, CD73, CD90, CD105, HLA-DR, HLA-ABC, immunocytochemistry for the proteins Nestina, ß-tubulin III, YAP and AMOT and RT-PCR for the NEFM and TUBB3 genes. Results Isolated cells demonstrated characteristics of MSCs. Those cells were differentiated in neural precursors, expressing the proteins Nestina and ß-tubulin III on immunocytochemistry and, the NEFM and TUBB3 genes in RT-PCR. Regarding the YAP and AMOT proteins, it was possible to observe the translocation of the YAP protein in response to the regulation of AMOT out of the cell nucleus, proving neurodifferentiation. Conclusions Human adipose-derived MSCs seeded in a natural biopolymer matrix were able to differentiate into neural precursors expressing characteristic neural markers without adding any neural growth factors or genetic induction.

scholarly journals 3D Printed SiOC(N) Ceramic Scaffolds for Bone Tissue Regeneration: Improved Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

2021 ◽  
Vol 22 (24) ◽  
pp. 13676
Author(s):  
Yuejiao Yang ◽  
Apoorv Kulkarni ◽  
Gian Domenico Soraru ◽  
Joshua M. Pearce ◽  
Antonella Motta
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
3D Printing ◽  
Pore Size ◽  
Osteogenic Differentiation ◽  
Bone Tissue ◽  
Low Cost ◽  
Human Mesenchymal Stem Cells ◽  
Bone Tissue Regeneration ◽  
Gene Expressions

Bone tissue engineering has developed significantly in recent years as there has been increasing demand for bone substitutes due to trauma, cancer, arthritis, and infections. The scaffolds for bone regeneration need to be mechanically stable and have a 3D architecture with interconnected pores. With the advances in additive manufacturing technology, these requirements can be fulfilled by 3D printing scaffolds with controlled geometry and porosity using a low-cost multistep process. The scaffolds, however, must also be bioactive to promote the environment for the cells to regenerate into bone tissue. To determine if a low-cost 3D printing method for bespoke SiOC(N) porous structures can regenerate bone, these structures were tested for osteointegration potential by using human mesenchymal stem cells (hMSCs). This includes checking the general biocompatibilities under the osteogenic differentiation environment (cell proliferation and metabolism). Moreover, cell morphology was observed by confocal microscopy, and gene expressions on typical osteogenic markers at different stages for bone formation were determined by real-time PCR. The results of the study showed the pore size of the scaffolds had a significant impact on differentiation. A certain range of pore size could stimulate osteogenic differentiation, thus promoting bone regrowth and regeneration.

Primordial germ cell differentiation of nuclear transfer embryonic stem cells using surface modified electroconductive scaffolds

2016 ◽  
Vol 53 (4) ◽  
pp. 371-380
Author(s):  
Tarlan Eslami-Arshaghi ◽  
Saeid Vakilian ◽  
Ehsan Seyedjafari ◽  
Abdolreza Ardeshirylajimi ◽  
Masoud Soleimani ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Differentiation ◽  
Embryonic Stem Cells ◽  
Germ Cell ◽  
Nuclear Transfer ◽  
Primordial Germ Cell ◽  
Embryonic Stem ◽  
Germ Cell Differentiation ◽  
Surface Modified

154 MOLECULAR CHARACTERIZATION OF CAT PRIMORDIAL GERM CELLS

2016 ◽  
Vol 28 (2) ◽  
pp. 207
Author(s):  
J. Galiguis ◽  
C. E. Pope ◽  
C. Dumas ◽  
G. Wang ◽  
R. A. MacLean ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Primordial Germ Cell ◽  
Embryonic Stem ◽  
Transcript Abundance ◽  
Domestic Cat ◽  
Sample Type

As precursors to germline stem cells and gametes, there are many potential applications for primordial germ cells (PGC). Primordial germ cell-like cells have been generated from mouse embryonic stem cells and induced pluripotent stem cells, which subsequently were used to produce functional spermatozoa, oocytes, and healthy offspring (Hayashi et al. 2012 Science 338(6109), 971–975). Applying this approach to generate sperm and oocytes of endangered species is an appealing prospect. Detection of molecular markers associated with PGC is essential to optimizing the process of PGC induction. In the current study, in vitro-derived domestic cat embryos were assessed at various developmental stages to characterise the expression of markers related to the specification process of cat PGC. In vivo-matured, IVF oocytes were cultured until Days 7, 9, and 12 post-insemination. Then, embryos were assessed by RT-qPCR to determine relative transcript abundance of the pluripotency markers NANOG, POU5F1, and SOX2; the epiblast marker DNMT3B; the primitive endoderm marker GATA4; the PGC marker PRDM14; and the germ cell marker VASA; RPS19 was used as the internal reference gene. To validate the qPCR results, fibroblasts served as the negative control cells, whereas spermatogonial stem cells (SSC) served as the positive control cells for GATA4, PRDM14, and VASA. Total mRNA were isolated using the Cells-to-cDNA™ II Kit (Ambion/Thermo Fisher Scientific, Waltham, MA, USA) from either pools of 2 to 6 embryos or ~25 000 fibroblasts/SSC. A minimum of 2 biological replicates for each sample type was analysed, with transcript abundance detected in 2 technical replicates by SYBR Green chemistry. Student’s t-tests were performed on the ΔCts for statistical analysis. PRDM14, specific to the germ cell lineage, was detected as early as Day 7, suggesting the presence of PGC precursor cells. Compared with their levels at Day 7, PRDM14 expression was 0.34-fold lower in SSC (P < 0.05), whereas expression of VASA and GATA4 were 1964-fold and 144-fold higher, respectively (P < 0.05). This seems to emphasise the relative importance of PRDM14 in pre-germ cell stages. In general, all genes analysed were up-regulated from Day 7 to Day 9. This up-regulation was statistically significant for SOX2 and GATA4 (P < 0.05). Relative to that at Day 9, all transcripts were relatively less abundant at Day 12 (P < 0.05 for NANOG, POU5F1, SOX2, DNMT3B, and PRDM14). The data suggest that PGC specification takes place near Day 9, with peak specification activity concluding by Day 12. Although much needs be explored about PGC specification in the cat before applying induction and in vitro germ cell production techniques, these findings represent the first step towards a new potential strategy for preserving endangered and threatened felids.

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.

scholarly journals Injectable Hydrogel Combined with Nucleus Pulposus-Derived Mesenchymal Stem Cells for the Treatment of Degenerative Intervertebral Disc in Rats

2019 ◽  
Vol 2019 ◽  
pp. 1-17 ◽  
Author(s):  
Feng Wang ◽  
Li-ping Nan ◽  
Shi-feng Zhou ◽  
Yang Liu ◽  
Ze-yu Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Intervertebral Disc ◽  
Nucleus Pulposus ◽  
Rat Model ◽  
Injectable Hydrogel ◽  
Cell Scaffold ◽  
Ivd Degeneration

Stem cell-based tissue engineering in treating intervertebral disc (IVD) degeneration is promising. An appropriate cell scaffold can maintain the viability and function of transplanted cells. Injectable hydrogel has the potential to be an appropriate cell scaffold as it can mimic the condition of the natural extracellular matrix (ECM) of nucleus pulposus (NP) and provide binding sites for cells. This study was aimed at investigating the effect of injectable hydrogel-loaded NP-derived mesenchymal stem cells (NPMSC) for the treatment of IVD degeneration (IDD) in rats. In this study, we selected injectable 3D-RGD peptide-modified polysaccharide hydrogel as a cell transplantation scaffold. In vitro, the biocompatibility, microstructure, and induced differentiation effect on NPMSC of the hydrogel were studied. In vivo, the regenerative effect of hydrogel-loaded NPMSC on degenerated NP in a rat model was evaluated. The results showed that NPMSC was biocompatible and able to induce differentiation in hydrogel in vivo. The disc height index (almost 87%) and MRI index (3313.83±227.79) of the hydrogel-loaded NPMSC group were significantly higher than those of other groups at 8 weeks after injection. Histological staining and immunofluorescence showed that the hydrogel-loaded NPMSC also partly restored the structure and ECM content of degenerated NP after 8 weeks. Moreover, the hydrogel could support long-term NPMSC survival and decrease cell apoptosis rate of the rat IVD. In conclusion, injectable hydrogel-loaded NPMSC transplantation can delay the level of IDD and promote the regeneration of the degenerative IVD in the rat model.

scholarly journals Primordial germ cell-like cells derived from canine adipose mesenchymal stem cells

2016 ◽  
Vol 49 (4) ◽  
pp. 503-511 ◽  
Author(s):  
Yudong Wei ◽  
Jia Fang ◽  
Shufang Cai ◽  
Changrong Lv ◽  
Shiqiang Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Germ Cell ◽  
Primordial Germ Cell ◽  
Adipose Mesenchymal Stem Cells

scholarly journals Bone marrow-derived mesenchymal stem cells combined with gonadotropin therapy restore postnatal oogenesis of chemo-ablated ovaries in rats via enhancing very small embryonic-like stem cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nesrine Ebrahim ◽  
Hajir A. Al Saihati ◽  
Amani Shaman ◽  
Arigue A. Dessouky ◽  
Ayman Samir Farid ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Histopathological Examination ◽  
Primordial Germ Cells ◽  
Average Density ◽  
Albino Rats ◽  
Epithelial Surface ◽  
Group V ◽  
Transcript Levels

Abstract Background Very small embryonic-like stem cells (VSELs) are a rare population within the ovarian epithelial surface. They contribute to postnatal oogenesis as they have the ability to generate immature oocytes and resist the chemotherapy. These cells express markers of pluripotent embryonic and primordial germ cells. Objective We aimed to explore the capability of VSELs in restoring the postnatal oogenesis of chemo-ablated rat ovaries treated with bone marrow-derived mesenchymal stem cells (BM-MSCs) combined with pregnant mare serum gonadotropin (PMSG). Methods Female albino rats were randomly assigned across five groups: I (control), II (chemo-ablation), III (chemo-ablation + PMSG), IV (chemo-ablation + MSCs), and V (chemo-ablation + PMSG + MSCs). Postnatal oogenesis was assessed through measurement of OCT4, OCT4A, Scp3, Mvh, Nobox, Dazl4, Nanog, Sca-1, FSHr, STRA8, Bax, miR143, and miR376a transcript levels using qRT-PCR. Expression of selected key proteins were established as further confirmation of transcript expression changes. Histopathological examination and ovarian hormonal assessment were determined. Results Group V displayed significant upregulation of all measured genes when compared with group II, III or IV. Protein expression confirmed the changes in transcript levels as group V displayed the highest average density in all targeted proteins. These results were confirmed histologically by the presence of cuboidal germinal epithelium, numerous primordial, unilaminar, and mature Graafian follicles in group V. Conclusion VSELs can restore the postnatal oogenesis in chemo-ablated ovaries treated by BM-MSCs combined with PMSG.

scholarly journals Natural Membrane Differentiates Human Adipose-Derived Mesenchymal Stem Cells to Neurospheres by Mechanotransduction Related to YAP and AMOT Proteins

Membranes ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 687
Author(s):  
Nathalia Barth de Oliveira ◽  
Ana Carolina Irioda ◽  
Priscila Elias Ferreira Stricker ◽  
Bassam Felipe Mogharbel ◽  
Nádia Nascimento da Rosa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Neuronal Differentiation ◽  
Rt Pcr ◽  
Isolated Cells ◽  
Neuronal Markers ◽  
Enzymatic Dissociation ◽  
Trilineage Differentiation ◽  
Good Cell

Adipose tissue-derived mesenchymal stem cells (ADMSCs) are promising candidates for regenerative medicine, as they have good cell yield and can differentiate into several cell lines. When induced to the neuronal differentiation, they form neurospheres composed of neural precursors (NPs) that can be an alternative in treating neurodegenerative diseases. This study aimed to characterize NPs from neurospheres obtained after seeding ADMSCs on a natural polyisoprene-based membrane. The ADMSCs were isolated from adipose tissue by enzymatic dissociation, were subjected to trilineage differentiation, and were characterized by flow cytometry for specific ADMSC surface markers. For neuronal differentiation, the cells were seeded on polystyrene flasks coated with the membrane and were characterized by immunocytochemistry and RT-PCR. The results demonstrated that the isolated cells showed characteristics of ADMSCs. At 15 to 25 days, ADMSCs seeded on the natural membrane developed neurospheres. Then, after dissociation, the cells demonstrated characteristic neuronal markers expressed on NPs: nestin, ß-III tubulin, GFAP, NeuN, and the YAP1/AMOT in the cytoplasm. In conclusion, it was demonstrated that this membrane differentiates the ADMSCs to NPs without any induction factors, and suggests that their differentiation mechanisms are related to mechanotransduction regulated by the YAP and AMOT proteins.

Isolation and culture of bone marrow mesenchymal stem cells from human fetus and their biological properties

2008 ◽  
Vol 5 (3) ◽  
pp. 237-244 ◽  
Author(s):  
Zhang Yi-Hua ◽  
Dou Zhong-Ying ◽  
Shen Wen-Zheng ◽  
Yang Chun-Rong ◽  
Gao Zhi-Min
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Calf Serum ◽  
Population Doubling ◽  
Basic Medium ◽  
Long Bones ◽  
Isolated Cells ◽  
Marrow Mesenchymal Stem Cells ◽  
Marrow Cells

AbstractThe population doubling number (70–80 times) of human fetal bone marrow mesenchymal stem cells (BMMSCs) is about two times higher than that (30–40 times) of adult BMMSCs, and their differentiation capacity is superior to that of their adult counterparts. In this study, BMMSCs were isolated from long bones of 2- to 3-month-old human abortuses by rinsing and selectively culturing whole marrow cells. Basic medium and serum concentration were optimized and growth curves plotted, both by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium bromide] reduction assay. Isolated cells were identified by flow cytometry and immunocytochemistry for their antigen markers. The biosafety of isolated cells was evaluated by karyotype analysis and a tumour-forming experiment. The results indicated that lengthwise scissoring of fetal long bones and rinsing of their marrow cells was practical and useful for recovery of BMMSCs from the investigated human abortuses. In this experiment, α-MEM (minimum essential medium, alpha medium)+20% FCS (fetal calf serum) was the best for in vitro culture of BMMSCs. The third-passage BMMSCs expressed Oct4, SSEA3 and SSEA4 besides the surface markers of their adult counterparts. The population doubling time of the BMMSCs of passage 6, 12 and 24 were 34, 36 and 40 h, respectively. Cells in all passages showed a diploid karyotype and formed no tumours in nude mice. The BMMSCs used in this study proved to be biologically safe and ideal seed cells for research on human tissue engineering and regeneration medicine.

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