210 COMPARATIVE CHARACTERIZATION OF MESENCHYMAL STEM CELLS DERIVED FROM MINIATURE PIG SYNOVIUM, SYNOVIAL FLUID AND BONE MARROW

2012 ◽  
Vol 24 (1) ◽  
pp. 217 ◽  
Author(s):  
W. J. Lee ◽  
G. H. Maeng ◽  
R. H. Jeon ◽  
G. J. Rho ◽  
S. L. Lee
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Blue Staining ◽  
Alcian Blue Staining ◽  
Miniature Pig ◽  
Rt Pcr ◽  
Alizarin Red ◽  
Differentiated Cells ◽  
Oil Red O Staining ◽  
Oil Red O

Mesenchymal stem cells (MSC) are a valuable cell source for cartilage tissue engineering because MSC derived from various tissues readily differentiate into skeletal cell and chondrogenic lineages. In this study, we compared the cellular characteristics of synovium (SN) and synovial fluid (SF)-derived MSC with bone marrow (BM)-derived MSC of miniature pig. Biopsies of SN and BM were collected and SF was flushed with saline solution from 6-month-old miniature pig (T-type, PWG Micro-pig, PWG Genetics Korea, South Korea). Cells were isolated from collected tissues and cultured in advanced-DMEM supplemented with 10% fetal bovine serum at 38.5°C in a humidified atmosphere of 5% CO2 in air. The cells were then evaluated for their expression of MSC-specific markers, including CD29, CD44 and CD90 using flow cytometry. The expression of early transcriptional factors, such as Oct3/4, Nanog and Sox2 was evaluated by immunocytochemical staining and reverse transcription-PCR (RT-PCR). Multilineage differentiation of MSC were induced under conditions conductive for osteogenic, adipogenic and chondrogenic lineages and then evaluated by von Kossa and Alizarin Red S staining, Oil red O staining and Alcian Blue staining, respectively. Differentiated cells were further analysed for the expression of lineage specific markers by RT-PCR. Statistical analysis was performed using one-way ANOVA by SPSS. The SN-, SF- and BM-MSC were observed to be positive for MSC specific markers, such as CD29 (99 ± 0.2, 96 ± 0.5 and 98 ± 0.2, respectively), CD44 (97 ± 0.3, 96 ± 0.6 and 98 ± 0.5, respectively) and CD90 (95 ± 0.5, 92 ± 0.2 and 96 ± 0.3, respectively); however, haematopoietic marker CD45 (2.0 ± 2.1, 3.0 ± 1.3 and 2.0 ± 3.0, respectively) was barely detected. In all MSC, early transcription factors (Oct3/4, Nanog and Sox2) were expressed by immnocytochemical staining and the transcripts were detected by RT-PCR. Following exposure to the specific differentiation medium, all these MSC were capable of differentiating into mesenchymal lineages, such as osteogenic, adiopogenic and chondrogenic as assessed by von Kossa and Alizarin Red S staining, Oil red O staining and and Alcian Blue staining, respectively. In addition, differentiated cells from all MSC expressed the marker genes specific to osteocytes (osteonectin, Runx2), adipocytes (aP2, PRAR-γ2) and chondrocytes (aggrecan, collagen type 2) by RT-PCR. The results of this study suggested that cells isolated from miniature pig articular tissues, such as SN and SF have similar characteristics of MSCs and their differentiation ability was comparable to BM-MSC. Hence, it is possible to establish MSCs from SN and SF as alternate sources during the biopsy of synovial tissues for arthritis diagnosis. Further studies are being carried out to evaluate their in vivo differentiation potential into chondrocytes. This study was supported by Rural Development Administration (grant No. 20110701-305-533-001-02-00) and National Research Foundation of Korea (grant No. 2011-0010252) of the Republic of Korea.

scholarly journals Lithium Chloride Modulates Adipogenesis and Osteogenesis of Human Bone Marrow-Derived Mesenchymal Stem Cells

2015 ◽  
Vol 37 (1) ◽  
pp. 143-152 ◽  
Author(s):  
Linjun Tang ◽  
Yu Chen ◽  
Fuxing Pei ◽  
Hui Zhang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Lithium Chloride ◽  
Psychiatric Medication ◽  
Alizarin Red ◽  
Treatment Of Osteoporosis ◽  
Candidate Drug ◽  
Oil Red O Staining ◽  
Oil Red O

Background/Aims: Lithium chloride (LiCl) has long been used as a psychiatric medication; however, its role in the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) remains largely unknown. The aim of this study is to explore the effect of LiCl on the differentiation of BMSCs. Methods: The roles of LiCl in osteogenic and adipogenic processes were observed using alizarin red staining and oil red O staining, respectively. The effects of LiCl on the Wnt and Hedgehog (Hh) pathways were investigated. Results: Our data showed that LiCl effectively promoted osteogenesis and inhibited adipogenesis by simultaneously affecting the Wnt and Hh pathways. Conclusion: These results suggest that LiCl influences the differentiation of BMSCs directly through the Wnt and Hh pathways and thus may be a candidate drug for the treatment of osteoporosis.

308 ISOLATION, DIFFERENTIATION, AND IMMUNOPHENOTYPIC CHARACTERIZATION OF MESENCHYMAL STEM CELLS DERIVED FROM EQUINE ADIPOSE TISSUE AND BONE MARROW

2011 ◽  
Vol 23 (1) ◽  
pp. 250
Author(s):  
E. Iacono ◽  
B. Merlo ◽  
A. Spadari ◽  
G. Mari ◽  
F. Ricci ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Cellular Therapy ◽  
Population Doubling ◽  
Blue Staining ◽  
Alcian Blue Staining ◽  
Alizarin Red ◽  
Minimum Criteria

Minimum criteria for the characterisation of human mesenchymal stem cells (MSC) are a) adhesion to the plastic when maintained under culture conditions; b) expression of CD105, CD73, and CD90, and no expression for CD45, CD34, and CD14; and c) differentiation into osteoblasts, adipocytes, and chondroblasts in vitro. One major difficulty in characterising equine MSC is the absence of specific monoclonal antibodies and evidence that certain markers from other species do not cross-react with the equine species. The aim of this work was to isolate, cultivate, differentiate, and conduct cellular characterisation of MSC derived from equine adipose tissue (AT) and bone marrow (BM). Adipose tissue collection was performed at the base of the horses’ tails, and BM was aspirated from the iliac crest. Mononuclear cell fraction was isolated and cultured as previously described by (Colleoni et al. 2009 Vet. Res. Commun. 33, 811–821). Chondrogenic, osteogenic, and adipogenic differentiation were performed in monolayer culture, and evidence for differentiation was made by morphological and cytological evaluations. For molecular characterisation, cells were treated with trypsin, washed with PBS, and fixed with Reagent 1 (Intraprep Kit, Beckman Coulter, Miami, FL, USA), following the manufacturer’s instructions. Samples, after washings, were incubated for 20 min at room temperature with CD105, CD90, CD44, CD45, CD34, CD14, and CD73 mAbs, directly conjugated to fluorescein isothiocyanate, PE, or APC (Beckman Coulter). Appropriate conjugate isotype controls were applied (Beckman Coulter). After staining, cells were washed twice with PBS, and fluorescence intensity was evaluated with a FC500 two-laser equipped cytometer (Beckman Coulter). Results were further analysed with the CXP dedicated program. Samples volumes were 68 ± 23.6 mL for BM and 5.6 ± 1.1 g for AT; in both AT and BM, the isolation rate was 100% (AT: 4/4; BM: 5/5). Undifferentiated cells were passaged up to 8 times for AT and 5 times for BM; population-doubling times (DT) were calculated, and data were analysed by ANOVA (Statistica for Windows, Stat Soft Inc., Tulsa, OK, USA). No significant differences (P > 0.05) were found between DT of all passages. The DT was greater (P < 0.05) for BM (3.2 ± 1.5) than for AT (1.3 ± 0.7). By passage 8, AT MSC underwent 37.3 ± 4.6 cell-doublings (CD); by passage 5, BM MSC underwent 26.2 ± 5.03 CD. Positive von Kossa and Alizarin Red staining confirmed osteogenesis. Alcian blue staining illustrated chondrogenesis, and positive Oil Red O staining suggested adipogenesis. The AT and BM MSC were positive for CD90, CD44, and CD105; all cell lines were negative for haematopoietic markers such as CD34, CD14, and CD45. Although marker CD73 expresses reaction in other studies involving MSC in different species, it did not cross-react with equine AT and BM MSC. Results obtained revealed the immunophenotypic characterisation of the surface of isolated and cultivated MSC, classifying these cells as a promising type of progenitor cells that can be applied in equine cellular therapy.

303 IN VITRO NEURONAL DIFFERENTIATION OF MESENCHYMAL STEM CELLS DERIVED FROM CANINE EAR SKIN

2011 ◽  
Vol 23 (1) ◽  
pp. 248
Author(s):  
J. H. Lee ◽  
Y. M. Lee ◽  
G. H. Maeng ◽  
S. L. Lee ◽  
G. J. Rho
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Immunohistochemical Staining ◽  
Transcriptional Factors ◽  
Surface Markers ◽  
Rt Pcr ◽  
Alizarin Red ◽  
Oil Red O

The canine has been a useful animal model for the study of fundamental mechanisms and the testing of new therapies for several human pathologies using mesenchymal stem cells (MSC). For preclinical applications, the most commonly used source of canine MSC is bone marrow. Because the amount of autologous bone marrow that can be obtained is limited, skin tissue could supply a noninvasive alternative with large quantities available for the establishment of MSC. In this study, we isolated canine MSC (cMSC) from ear skin and evaluated the expression of transcriptional factors and in vitro differentiation into multiple mesenchymal lineages. The cMSC isolated from the ear skin of a female beagle dog (6 years old) were cultured in advanced-DMEM/F12 (1:1, v/v) supplemented with 10% serum replacement at 37°C, 5% CO2 in a humidified atmosphere. The cMSC at passage 3 were analysed for expression of surface markers (CD44, CD90, and CD105) and transcriptional factors (Oct-4, Nanog, and Sox2) using flow cytometry, immunohistochemical staining and RT-PCR, respectively. Differentiations into adipocytes and osteocytes of cMSC were carried out under controlled conditions for 2 and 4 weeks and evaluated by staining (Oil Red O, von Kossa and Alizarin Red S, respectively). The cMSC were induced to differentiate into neural cells in the controlled condition for 6 h. Neuronal differentiated cMSC were evaluated by immunohistochemical staining, RT-PCR, and Western blot of specific markers of neuron, such as Î2-tubulin, microtubule associated protein (MAP-2), neuronfilament M (NF-M), nerve growth factor (NGF), and nestin. The MSC surface markers such as CD44, 90, and 105 were highly detected, and transcriptional factors (Oct-4, Nanog, and Sox2) were expressed in cMSC. Adipocyte induced cells were positive by staining with Oil Red O, and osteocytes were stained by von Kossa and Alizarin Red S. Neuronal specific markers such as Î2-tubulin, MAP-2, NF-M, NGF, and nestin were expressed in the neuron induced cMSC. In conclusion, canine ear-skin-derived MSC have the capacity for differentiation into multiple lineages and have a confirmed great capability for neuronal differentiation. Hence, canine ear skin tissue could be considered a source for applications of MSC for neuronal regeneration therapy of canine and a preclinical research model for human. This work was supported by Grant No. 2007031034040 from Bio-organ and Grant No. 200908FHT010204005 from Biogreen21.

296 IN VITRO DIFFERENTIATION OF PORCINE BONE MARROW-DERIVED MESENCHYMAL STEM CELLS INTO HEPATOCYTE-LIKE CELLS

2013 ◽  
Vol 25 (1) ◽  
pp. 295
Author(s):  
B. Mohana Kumar ◽  
W. J. Lee ◽  
Y. M. Lee ◽  
R. Patil ◽  
S. L. Lee ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Class Ii ◽  
In Vitro Differentiation ◽  
Rt Pcr ◽  
Hepatic Differentiation ◽  
Alizarin Red ◽  
Differentiation Potential

Mesenchymal stem cells (MSC) are isolated from bone marrow or other tissues, and have properties of self renewal and multilineage differentiation ability. The current study investigated the in vitro differentiation potential of porcine bone marrow derived MSCs into hepatocyte-like cells. The MSC were isolated from the bone marrow of adult miniature pigs (7 months old, T-type, PWG Micro-pig®, PWG Genetics, Seoul, Korea) and adherent cells with fibroblast-like morphology were cultured on plastic. Isolated MSCs were positive for CD29, CD44, CD73, CD90, and vimentin, and negative for CD34, CD45, major histocompatibility complex-class II (MHC-class II), and swine leukocyte antigen-DR (SLA-DR) by flow cytometry analysis. Further, trilineage differentiation of MSC into osteocytes (alkaline phosphatase, von Kossa and Alizarin red), adipocytes (Oil Red O), and chondrocytes (Alcian blue) was confirmed. Differentiation of MSC into hepatocyte-like cells was induced with sequential supplementation of growth factors, cytokines, and hormones for 21 days as described previously (Taléns-Visconti et al. 2006 World J. Gastroenterol. 12, 5834–5845). Morphological analysis, expression of liver-specific markers, and functional assays were performed to evaluate the hepatic differentiation of MSC. Under hepatogenic conditions, MSC acquired cuboidal morphology with cytoplasmic granules. These hepatocyte-like cells expressed α-fetoprotein (AFP), albumin (ALB), cytokeratin 18 (CK18), cytochrome P450 7A1 (CYP7A1), and hepatocyte nuclear factor 1 (HNF-1) markers by immunofluorescence assay. In addition, the expression of selected markers was demonstrated by Western blotting analysis. In accordance with these features, RT-PCR revealed transcripts of AFP, ALB, CK18, CYP7A1, and HNF-1α. Further, the relative expression levels of these transcripts were analysed by quantitative RT-PCR after normalizing to the expression of the endogenous control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data were analysed statistically by one-way ANOVA using PASW statistics 18 (SPSS Inc., Chicago, IL, USA), and significance was considered at P < 0.05. The results showed that the relative expressions of selected marker genes in hepatocyte-like cells were significantly increased compared with that in untreated MSC. The generated hepatocyte-like cells showed glycogen storage as analysed by periodic acid-Schiff (PAS) staining. Moreover, the induced cells produced urea at Day 21 of culture compared with control MSC. In conclusion, our results indicate the potential of porcine MSC to differentiate in vitro into hepatocyte-like cells. Further studies on the functional properties of hepatocyte-like cells are needed to use porcine MSC as an ideal source for liver cell therapy and preclinical drug evaluation. This work was supported by Basic Science Research Program through the National Research Foundation (NRF), funded by the Ministry of Education, Science and Technology (2010-0010528) and the Next-Generation BioGreen 21 Program (No. PJ009021), Rural Development Administration, Republic of Korea.

scholarly journals Endometrial Mesenchymal Stem Cells Isolated from Menstrual Blood by Adherence

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Xue Du ◽  
Qing Yuan ◽  
Ye Qu ◽  
Yuan Zhou ◽  
Jia Bei
Keyword(s):  
Growth Factor ◽  
Alcian Blue ◽  
Gradient Centrifugation ◽  
Menstrual Blood ◽  
Blue Staining ◽  
Enzyme Linked Immunosorbent Assay ◽  
Alcian Blue Staining ◽  
Promising Alternative ◽  
Oil Red O Staining ◽  
Oil Red O

Objective. To find a convenient and efficient way to isolate MSCs from human menstrual blood and to investigate their biological characteristics, proliferative capacity, and secretion levels.Methods. MSCs were isolated from menstrual blood of 3 healthy women using adherence. Cell immunological phenotype was examined by flow cytometry; the adipogenic, osteogenic, and chondrogenic differentiation of MSCs was examined by Oil-Red-O staining, ALP staining, and Alcian Blue staining, respectively; and the secretion of cytokines, including vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1), was detected using enzyme-linked immunosorbent assay.Results. MB-MSCs were successfully isolated from human menstrual blood using adherence. They were positive for CD73, CD105, CD29, and CD44, but negative for CD31 and CD45. The differentiated MB-MSCs were positive for ALP staining, Oil-Red-O staining, and Alcian Blue staining. In addition, they could secrete antiapoptotic cytokines, such as VEGF, IGF-1, and HGF.Conclusion. It is feasible to isolate MSCs from human menstrual blood, thus avoiding invasive procedures and ethical controversies. Adherence could be a promising alternative to the density gradient centrifugation for the isolation of MSCs from menstrual blood.

scholarly journals Knockdown of CDC20 Promotes Adipogenesis of Bone Marrow-derived Stem Cells

2021 ◽  
Author(s):  
Yangge Du ◽  
Yunsong Liu ◽  
Yongsheng Zhou ◽  
Ping Zhang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Adipose Tissue ◽  
Western Blot ◽  
Adipogenic Differentiation ◽  
Conditional Knockout ◽  
Qrt Pcr ◽  
Oil Red O Staining ◽  
Oil Red O

Abstract Background: Bone is a rigid organ that provides support and physical protection to vital organs of the body. Several bone loss disorders are commonly associated with increased bone marrow adipose tissue. Bone marrow mesenchymal stromal/stem cells (BMSCs) are multipotent progenitors differentiating into osteoblasts, adipocytes, and chondrocytes. CDC20 is a co-activator of APC/C, required for full ubiquitin ligase activity. In our previous study, CDC20 promoted the osteogenic commitment of BMSCs and Cdc20 conditional knockout mice suggested a decline in bone mass. In this study, we investigated the function of CDC20 in the adipogenic differentiation of BMSCs and provided a new clue between adipogenesis and osteogenesis. Methods: Lentivirus containing CDC20 shRNA was used for CDC20 knockdown in hBMSCs. Primary mBMSCs were isolated from Cdc20f/f and Sp7-Cre;Cdc20f/f mice. Adipogenesis was examined by qRT-PCR and western blot analysis of adipogenic regulators, Oil Red O staining and transplantation into nude mice. The CDC20 knockout efficiency was determined through immunochemistry, qRT-PCR and western blot of bone marrow. Accumulation of adiposity was measured through histology and staining of bone sections. Results: CDC20 expression in hBMSCs was significantly decreased during adipogenic differentiation. Knockdown of CDC20 enhanced adipogenic differentiation of hBMSCs in vitro. CDC20-knockdown hBMSCs showed more adipose tissue–like constructs in H&E staining and Oil Red O staining. Sp7-Cre;Cdc20f/f mice presented increased adipocytes in bone marrow compared with control mice. mBMSCs from Sp7-Cre;Cdc20f/f mice exerted upregulated adipogenic differentiation. Conclusions: Our findings showed that knockdown of CDC20 enhanced adipogenesis of h(m)BMSCs in vitro and in vivo. Overall, CDC20 regulated both adipogenesis and osteogenesis of BMSCs, and may lead to the development of new therapeutic target for “fatty bone” and osteoporosis.

In vitro differentiation of chicken spermatogonial stem cells into adipocytes

2008 ◽  
Vol 5 (3) ◽  
pp. 263-268
Author(s):  
Yu Fei ◽  
Ge Jian-Hui ◽  
Ni Li-Gang ◽  
He Xian-Hong ◽  
Xu Qi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Spermatogonial Stem Cells ◽  
In Vitro Differentiation ◽  
Differentiated Cells ◽  
Ppar Γ ◽  
Combined Use ◽  
Oil Red O Staining ◽  
Oil Red O ◽  
Better Than

AbstractSpermatogonial stem cells (SSCs), which were isolated from chicken (Gallus gallus) embryo testes 16 days after laying, were cultured, subcultured, and induced into adipocytes in vitro. The differentiated cells were identified by oil red-O staining. Dexamethasone, insulin and 3-isobutyl-1-methylxanthine (IBMX) were tested for their induction potential. About 7–21 days after induction, SSCs differentiated into adipocytes, and the resulting adipocytes strongly expressed peroxisome proliferation activation receptor-γ (PPAR-γ). The assay outcome showed that an optimal treatment consisted of dexamethasone, insulin and IBMX application for 3 days and insulin for 1 day (3 cycles), then insulin for 21 days. The differentiation ratio was 85%, better than the combined use of dexamethasone, insulin and IBMX (P<0.01). However, the combination of the three derivatives triggered a stronger induction than any of them used alone (P<0.01). This study has demonstrated the potential of chicken embryonic SSCs to differentiate in vitro into adipocytes.

Mycophenolic Acid Inhibits the Proliferation and Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells by Guanosine Depletion.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1454-1454 ◽  
Author(s):  
Weijie Cao ◽  
Lizhen Liu ◽  
Xiaoyu Lai ◽  
Xiaohong Yu ◽  
He Huang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Adipogenic Differentiation ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Dependent Manner ◽  
Oil Red O Staining ◽  
Oil Red O

Abstract Abstract 1454 Poster Board I-477 Introduction Mycophenolate mofetil is now widely used in transplantation as a potent immunosuppressant, whose active metabolite is mycophenolic acid (MPA). MPA inhibits de novo purine biosynthesis by reversible, noncompetitive inhibition of inosine monophosphate dehydrogenase (IMPDH). The inhibition of IMPDH in lymphocytes reduces intracellular guanine nucleotide pools, thus arrests lymphocytes proliferation. Recently investigators reported the antiproliferative effects of MPA on fibroblasts, smooth muscle cells and endothelial cells, but there is no reports of the effects of MPA on human bone marrow-derived mesenchymal stem cells (MSCs). Here we examined the effects of MPA on the proliferation and differentiation of human bone marrow-derived mesenchymal stem cells. Methods Bone marrow aspirates were obtained from healthy volunteers after informed consent, and MSCs were expanded from bone marrow mononuclear cells by discarding non-adherent cells. For proliferation and survival assays, MSCs were treated with MPA at the concentration of 1μM, 10μM, 50μM, and 100μM. Cell proliferation was analyzed using CCK-8 method (Dojindo). Cell viability was assessed by trypan blue exclusion. Apoptosis was detected by PI/Annexin V assay kit (Invitrogen). To assess the effects of MPA on MSCs differentiation, osteogenic differentiation and adipogenic differentiation were induced in the presence of MPA. For the detection of osteogenic differentiation, the deposited minerals was stained with silver by the method of von Kossa and Ca2+ contents was quantified with calcium colorimetric assay kit (Biovision). Adipogenic differentiation was analyzed by Oil Red O staining and Oil Red O staining extraction. Results In the range of 1μM to 100μM, MPA caused a significant subdued proliferation rate of MSCs in a concentration- and time-dependent manner. After 7d of incubation with MPA at the concentration of 1μM, 10μM, 50μM, and 100μM, the proliferation rate was reduced to 65.33±11.03%, 24±3.74%, 15.33±4.03%, and 15.33±6.94% respectively (P<0.01). Adding guanosine (100μM) to the culture restored the proliferation rate (P<0.01) indicating that MPA exerted antiproliferative effects by guanosine depletion. Trypan blue staining showed that there was no statistically significant difference in the ratio of living cells between MPA treated cells and the control group (P>0.05), and PI/Annexin V staining showed no apoptosis induce by MPA. Von Kossa stainnging indicated that treatment with MPA reduced Ca2+ deposition during osteogenic differentiation of MSCs, and Ca2+ quantification further confirmed that MPA inhibited osteogenic differentiation in a concentration-dependent manner. Ca2+ quantification was 78.43±12.79 μg/well and 22.8±6.58 μg/well respectively at the concentration of 10μM and 100μM of MPA, which were significantly lower than the control group(118.33±12.50ug/well, P<0.05). Oil Red O staining and Quantification of lipid contents showed that MPA had no effect on lipid production during adipogenic differentiation. Conclusion Our study demonstrated that MPA inhibited the proliferation of MSCs by guanosine depletion, and also inhibited the osteogenic differentiation in a concentration-dependent manner. However, MPA had no impact on adipogenic differentiation in vitro. Disclosures No relevant conflicts of interest to declare.

scholarly journals The effect of aging on the biological and immunological characteristics of periodontal ligament stem cells

2020 ◽  
Author(s):  
Xiaoyu Li ◽  
Bowen Zhang ◽  
Hong Wang ◽  
Xiaolu Zhao ◽  
Zijie Zhang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Periodontal Ligament ◽  
Chondrogenic Differentiation ◽  
Annexin V ◽  
Cell Counting ◽  
Blue Staining ◽  
Alcian Blue Staining ◽  
Periodontal Ligament Stem Cells ◽  
Alizarin Red ◽  
Differentiation Potential

Abstract Background: Periodontal ligament stem cells (PDLSCs) have many applications in the field of cytotherapy, tissue engineering, and regenerative medicine. However, the effect of age on the biological and immunological characteristics of PDLSCs remains unclear. Methods: In this study, we compared PDLSCs isolated from young and adult individuals. PDLSCs proliferation was analyzed by Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) staining, and apoptosis level was detected by Annexin V-PE/7-ADD staining. PDLSCs osteogenic/adipogenic/chondrogenic differentiation potentials were assessed by alkaline phosphatase (ALP), Alizarin Red, Oil Red O, Alcian Blue staining and related quantitative analysis. PDLSCs immunosuppressive capacity was determined by EdU and Annexin V-PE/7-ADD staining. To explore its underlying mechanism, microarray, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), and western blot analyses were performed to detect differentially expressed genes and proteins in PDLSCs. Results: Our results demonstrated that with aging, the proliferation and osteogenic/adipogenic/chondrogenic differentiation potential of PDLSCs decreased, whereas apoptosis of PDLSCs increased. Moreover, the immunosuppressive ability of PDLSCs decreased with aging. Compared with PDLSCs from young subjects, analysis of mRNA expression revealed an upregulation of CCND3 and RC3H2, and a downregulation of Runx2, ALP, COL1A1, PPARγ2, CXCL12, FKBP1A, FKBP1B, NCSTN, P2RX7, PPP3CB, RIPK2, SLC11A1, and TP53 in those from adult individuals. Furthermore, protein expression levels of Runx2, ALP, COL1A1, and PPARγ2 in the adult group was decreased, whereas that of CCND3 increased. Conclusions: Taken together, aging influences biological and immunological characteristics of PDLSCs, and thus it is more appropriate to utilized PDLSCs from young individuals for tissue regeneration, post-aging treatment, and allotransplantation.

168 CHARACTERIZATION AND DIFFERENTIATION INTO OOCYTE-LIKE CELL MASSES OF PORCINE MESENCHYMAL STEM CELLS DERIVED FROM OVARIAN THECA CELLS

2011 ◽  
Vol 23 (1) ◽  
pp. 186 ◽  
Author(s):  
Y. M. Lee ◽  
B. Mohana Kumar ◽  
S. W. Kim ◽  
S. L. Lee ◽  
G. J. Rho
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Transcriptional Factors ◽  
Immunocytochemical Staining ◽  
Blue Staining ◽  
Specific Marker ◽  
Rt Pcr ◽  
Theca Cells ◽  
Differentiated Cells

Recent findings have shown that ovaries after birth have germ line stem cells, which were considered as an alternative for the production of an animal model. The present study was therefore aimed to characterise ovarian theca cells and generate oocyte-like cell masses in vitro in porcine. Theca cells isolated from ovarian follicle were cultured in A-DMEM supplemented with 10% FBS at 38.5°C in a humidified atmosphere of 5% CO2 in air. The cells were evaluated the expression of transcriptional factors (Oct3/4, Nanog, and Sox2) by immunocytochemical staining and RT-PCR, and followed by differentiated into osteocytes, adipocytes, and chondrocytes under controlled conditions. Differentiation of multiple mesenchymal lineages was confirmed by RT-PCR and specific marker staining. Differentiated cells into osteocytes, adipocytes, and chondrocytes were characterised by von Kossa and Alizarin Red staining, Oil red O staining, and Alcian Blue staining, respectively. The specific genes of osteocytes (Osteonectin, Osteocalcin and Runx2) and adipocytes (aP2) were analysed by RT-PCR. In vitro oogenesis was induced in DMEM/F12 by the previously described method (Dyce et al. 2006) for 48 days. Expression of transcriptional factors (Oct4, Sox2, and Nanog) and oocyte-specific markers (c-Mos and GDF9b) was analysed by RT-PCR in these differentiated cells. At 48 days of differentiation, the oocyte-like cell masses were further cultured in TCM-199 supplemented with 0.5 μL mL–1 FSH and 0.5 μL mL–1 LH for 15 days. Induced cells were morphologically observed following Hoechst 33342. Expression of Oct3/4 was analysed by immunocytochemical staining in these cells. Among the transcriptional factors, only Sox2 was detected by immunocytochemical staining and RT-PCR in the theca cells. Differentiation to osteocytes, adipocyte, and chondrocytes was confirmed by specific-marker staining and gene expression by RT-PCR, respectively. The morphology of oocyte-like cell masses was distinct by 40 days of differentiation. Granulosa or cumulus-like cells were distributed through the whole surface of oocyte-like cell masses. Transcriptional factors, c-Mos, and GDF9b were detected in the cell masses by RT-PCR. After being transferred oocyte-like cell masses to TCM-199, zona pellucida-like structure was formed around the edge of the cell mass. After 15 days of culture in TCM-199, the morphology of cells was changed into blastocyst-like structure, which surrounded cumulus-like cells. Oct3/4 was expressed by immunocytochemical staining in a blastocyst-like structure. These observations demonstrated that ovarian theca cells have similar characteristics to mesenchymal stem cells in view of multilineage differentiation. Theca cells can be differentiated into oocyte-like cell masses, which expressed oocyte-specific markers. These cell masses were further developed to a blastocyst-like structure, which expressed Oct3/4. Further studies are required to evaluate in vivo differentiation to oocyte-like cells. This work was supported by Grant No. 200908FHT010204005 from Biogreen21 and Grant No. 2007031034040 from Bio-organ.

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