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 Melatonin Reverses the Loss of Stemness Induced by TNF-α in Human Bone Marrow Mesenchymal Stem Cells through Upregulation of YAP Expression

2019 ◽  
Vol 2019 ◽  
pp. 1-16 ◽  
Author(s):  
Xudong Wang ◽  
Tongzhou Liang ◽  
Jincheng Qiu ◽  
Xianjian Qiu ◽  
Bo Gao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Molecular Mechanisms ◽  
Inflammatory Factor ◽  
Cell Transplant ◽  
Alizarin Red ◽  
Marrow Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are promising candidates for tissue regeneration and disease treatment. However, long-term in vitro culture results in loss of MSC stemness. The inflammation that occurs at stem cell transplant sites (such as that resulting from TNF-α) is a contributing factor for stem cell treatment failure. Currently, there is little evidence regarding the protective role of melatonin with regard to the negative effects of TNF-α on the stemness of MSCs. In this study, we report a melatonin-based method to reduce the inflammatory effects on the stemness of bone marrow mesenchymal stem cells (BMMSCs). The results of colony formation assays, Alizarin red staining, western blotting, and reverse transcription-polymerase chain reactions suggest that melatonin can reverse the inflammatory damage caused by TNF-α treatment in the third, seventh, and tenth generations of primary BMMSCs (vs. control and the TNF-α-treated group). Meanwhile, a detailed analysis of the molecular mechanisms showed that the melatonin receptor and YAP signaling pathway are closely related to the role that melatonin plays in negative inflammatory effects against BMMSCs. In addition, in vivo experiments showed that melatonin could reverse the damage caused by TNF-α on bone regeneration by BMMSCs in nude mice. Overall, our results suggest that melatonin can reverse the loss of stemness caused by inflammatory factor TNF-α in BMMSCs. Our results also provide a practical strategy for the application of BMMSCs in tissue engineering and cell therapy.

306 IN VITRO CARDIOMYOGENIC AND NEUROGENIC DIFFERENTIATION OF MINIPIG BONE MARROW MESENCHYMAL STEM CELLS

2011 ◽  
Vol 23 (1) ◽  
pp. 249
Author(s):  
B. Mohana Kumar ◽  
T. H. Kim ◽  
Y. M. Lee ◽  
G. H. Maeng ◽  
B. G. Jeon ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Cardiac Troponin ◽  
Troponin T ◽  
Cardiac Troponin T ◽  
Rt Pcr ◽  
Neurogenic Differentiation ◽  
Cardiac Actin

Differentiation of mesenchymal stem cells (MSC) into specialised cells in vitro before transplantation may improve the engraftment efficiency of the transplanted cells as well as the safety and efficacy of treatment. To understand the differentiation process and the functional identities of cells in an animal model, we examined the in vitro differentiation capacity of porcine MSC (3–6 passage) into cardiomyocyte-like and neuron-like cells. The MSC isolated from the bone marrow of postnatal miniature piglets [T-type, PWG Micro-pig (R), PWG Genetics, Korea] exhibited a typical fibroblast-like morphology and expressed the specific markers, such as CD29, CD44, and CD90. After 21 days of culture in induction media, MSC revealed the appropriate phenotype of osteocytes (von Kossa and Alizarin red), adipocytes (Oil red O), and chondrocytes (Alcian blue). Ther MSC were further induced into cardiomyogenic and neurogenic differentiation following the protocols described earlier (Tomita et al. 2002 J. Thorac. Cardiovasc. Surg. 123, 1132–1140) and (Woodbury et al. 2002 J. Neurosci. Res. 96, 908–917), respectively, with minor modifications. Expression of lineage-specific markers was evaluated by immunocytochemistry, and RT-PCR and quantitative PCR (RT-qPCR). For cardiomyogenic differentiation, MSC were stimulated with 10 μM 5-azacytidine for 24 h, 3 days, or 7 days, and the cells were maintained in culture for 21 days. Upon induction, MSC exhibited elongated and stick-like morphology with extended cytoplasmic processes, and toward the end of culture, cells formed aggregates and myotube-like structures. Immunostaining was positive for the markers of cardiomyocyte-like cells, such as α-smooth muscle actin, cardiac troponin T, desmin, and α-cardiac actin. The RT-PCR and RT-qPCR analysis showed the expression and a time dependent up-regulation of cardiac troponin T, desmin, α-cardiac actin, and β-myosin heavy chain genes. Following induction with neuronal-specific media for 3 days, above 80% of MSC acquired the morphology of neuron-like cells with bi- or multipolar cell processes forming a network-like structure. Induced cells with neuronal phenotype were positively stained for nestin, neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), and neurofilament-M (NF-M). The expression of neural transcripts, such as nestin, GFAP, and NF-M, was further confirmed by RT-PCR and RT-qPCR. In conclusion, our results showed the potential of porcine MSC to differentiate in vitro into cardiomyocyte-like and neuron-like cells, thus offering a useful model for studying their functional and molecular properties before transplantation. 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 BioGreen 21 (20070301034040), Republic of Korea.

300 ESTABLISHMENT AND CHARACTERIZATION OF RAT MESENCHYMAL STEM CELLS

2011 ◽  
Vol 23 (1) ◽  
pp. 247
Author(s):  
T. H. Kim ◽  
B. G. Jeon ◽  
S. L. Lee ◽  
G. J. Rho
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Transcriptional Factors ◽  
Skin Tissue ◽  
Rt Pcr ◽  
Alternative Source ◽  
Differentiated Cells ◽  
Fetal Bovine

Mesenchymal stem cells (MSC) are regarded as an attractive source for tissue engineering and regeneration, and bone marrow extract has been commonly used as a source of pluripotent MSC. However, skin tissue has recently been identified as a convenient alternative source of MSC. The present study was focused on the effect of characterised MSC derived from rat on expression of early transcriptional factors, alkaline phosphate (AP) activity, and in vitro differentiation into selected cell lineages. The MSC were isolated from 8-week-old s.d. rat’s ear skin and cultured in advanced DMEM supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere of 5% CO2 in air. To evaluate AP activity, cells were fixed with 3.7% formaldehyde solution and stained with Western Blue® (Promega, Madison, WI, USA). Expressions of early transcriptional factors (Oct-4, Sox2, and Nanog) were evaluated by RT-PCR. Differentiation into distinct mesenchymal lineages such as adipogenic, osteogenic, and neuron was done by following previously described protocols and assessed by lineage-specific stains. The specific genes in the osteocytes (osteocalcin, osteonectin, osteopontin, and Runx2), adipocytes (pparγ2, adiponectin, and aP2) or neuron (nestin, neurogenin 1, β-tublin, and nerve growth factor) were characterised by RT-PCR. The MSC were positive for AP activity and expressed Oct-4, Sox2, and Nanog. Following induction, MSC were successfully differentiated into adipocytes, osteocytes, and neurons. As adipocytes markers, aP2, pparγ2, and adiponectin were strongly detected in the adipocyte induced cells. Osteonectin, osteocalcin, Runx2, and osteopontin were expressed in the adipocyte induced cells. Futhermore, neuron-specific markers were clearly expressed in the neuronal differentiated cells. In conclusion, MSC have the capability of differentiation into multilineages including adipocytes, osteocytes, and neurons under the specific induction conditions. Skin tissue in rat can serve as an easily accessible and expandable alternative source for MSC harvesting and preclinical applications using an animal model. This work was supported by Grant No. 2007031034040 from Bio-organ and 200908FHT010204005 from Biogreen21, Republic of Korea.

Differentiation of Mesenchymal Stem Cells from Rat Bone Marrow into Dopaminergic Neuron-Like Cells In Vitro.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4067-4067
Author(s):  
Li Chen ◽  
Dongmei He ◽  
Yuan Zhang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Growth Factor ◽  
Dopaminergic Neuron ◽  
Bone Marrow Mscs ◽  
Rt Pcr ◽  
Promising Source ◽  
Rat Bone

Abstract Mesenchymal stem cells (MSC) from bone marrow cavity are multipotent cells. Their primary function is to support the growth and differentiation of hematologic progenitors. MSCs have been shown to differentiate into a variety of cell types including: bone, adipocytes, cartilage, neuron-like, and muscle-like cells. This project aimed to induce MSCs from rat bone marrow into mature dopamine secreting cells. MSCs were isolated from rat bone marrow, cultured and passaged. After propagating for three generations in vitro culture, MSCs were induced by epidermal growth factor, basic fibroblast growth factor and retinoic acid. After induction, morphologic change was examined by light microscope. NSE,MAP-2a, b and tyrosine hydroxylase (TH) was examined by immunocytochemistry. The related genes of the differentiated neurons, such as Nurr-1, nestin, mash-1,DR2-L,AADC and TH were detected by RT-PCR. After MSCs were inducted for 7 days,14 days and 21 days, dopamine production and release in the extract and medium of dopaminergic-induced cultured cells was assayed by dopamine ELISA. After 14 days of induction, MSC showed neuron-like morphologic changes and expressed NSE, MAP-2a, b and TH. RT-PCR. showed that these induced cells expressed nerves stem cells gene Nestin,Nurr-1 and dopamine nerves gene mash-1,DR2-L,AADC,TH. Most importantly, dopamine ELISA analysis showed the evidence of dopamine release in the extract and medium of dopaminergic-induced clonal MSCs. The results suggest that bone marrow MSCs from rat can be induced to differentiate into dopaminergic neuron-like cells in vitro. Bone marrow MSCs will provide a promising source of neural progenitor cells and may be a favorable candidate for cellular therapy of Parkinson’s disease.

206 GENERATION OF HUMAN INDUCED PLURIPOTENT STEM CELLS FROM DENTAL PULP-DERIVED MESENCHYMAL STEM CELLS

2012 ◽  
Vol 24 (1) ◽  
pp. 215 ◽  
Author(s):  
J. H. Lee ◽  
Y. M. Lee ◽  
G. H. Maeng ◽  
R. H. Jeon ◽  
T. H. Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dental Pulp ◽  
Ips Cells ◽  
Retroviral Vector ◽  
Transcriptional Factors ◽  
Alizarin Red ◽  
Differentiation Potential ◽  
Induced Pluripotent ◽  
Oil Red O

Induced pluripotent stem (iPS) cells are somatic cells that have been reprogrammed to a pluripotent state and a great source for regenerative medicine. Several types of human somatic and adult stem cells have been reprogrammed into iPS cells, including mesenchymal stem cells (MSC). Recently, human dental pulp has been considered as a valuable alternative source of MSC (hDP-MSC) with excellent proliferation capacity and multilineage differentiation potential. In this study, our objective was to establish iPS cells from hDP-MSC and evaluate the expression of transcriptional factors and in vitro differentiation potential into mesenchymal lineages. The hMSC were isolated from the dental pulp of male donor (∼18 years old) were cultured in advanced-DMEM supplemented with 10% fetal bovine serum at 37°C, 5% CO2 in a humidified atmosphere. The hDP-MSC at passage 3 were analysed for the expression of MSC-specific surface markers (CD44 and CD90) using flow cytometry and transcriptional factors (Oct4, Nanog and Sox2) by immunofluorescence staining and reverse transcription-polymerase chain reaction (RT-PCR). Differentiation into adipocytes and osteocytes of hDP-MSC was carried out under specific conditions for 2 and 4 weeks, respectively and assessed by cytochemical staining (Oil red O, von Kossa and Alizarin Red S, respectively). iPS cells were generated from hDP-MSC at passage 3 by using pMXs retroviral vector (Addgene, Cambridge, MA, USA) containing cDNA of c-Myc, Klf4, Nanog and Sox2. The iPS cells were evaluated for alkaline phosphatase (AP) activity, expression of human embryonic stem cells (hESC) markers (Rex1, Nanog, Oct4, SSEA-1 and TRA-160) by immunostaining. Isolated hDP-MSC expressed surface markers, such as CD44 and CD90 (86% and 93%, respectively) by flow cytometry and positively stained for transcriptional factors (Oct4, Nanog and Sox2) by immunofluorescence. Further, the cells were capable of differentiating in vitro into adipocytes and osteocytes as demonstrated by Oil red O and von Kossa and Alizarin red S staining, respectively. The iPS cells generated from hDP-MSC were positive for AP staining and clearly expressed the markers specific to hESC, including Rex1, Nanog, Oct4, SSEA-1 and TRA-160. In conclusion, hMSC derived from dental pulp could be successfully reprogrammed into iPS cells by retroviral vector systems and the generated iPS cells shared the similar characteristics of hESC. Therefore, hDP-MSC might be an ideal alternative cell source to derive autologous iPS cells for therapeutic applications. This work was supported by Grant No. 2007031034040 from Bio-organ and Grant No. 200908FHT010204005 from Biogreen21.

scholarly journals An Improved Harvest andin VitroExpansion Protocol for Murine Bone Marrow-Derived Mesenchymal Stem Cells

2010 ◽  
Vol 2010 ◽  
pp. 1-10 ◽  
Author(s):  
Song Xu ◽  
Ann De Becker ◽  
Ben Van Camp ◽  
Karin Vanderkerken ◽  
Ivan Van Riet
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Surface Markers ◽  
Differentiation Potential ◽  
Murine Bone Marrow ◽  
Expansion Time ◽  
Expansion Protocol ◽  
In Vitro Expansion

Compared to bone marrow (BM) derived mesenchymal stem cells (MSCs) from human origin or from other species, the in vitro expansion and purification of murine MSCs (mMSCs) is much more difficult because of the low MSC yield and the unwanted growth of non-MSCs in the in vitro expansion cultures. We describe a modified protocol to isolate and expand murine BM derived MSCs based on the combination of mechanical crushing and collagenase digestion at the moment of harvest, followed by an immunodepletion step using microbeads coated with CD11b, CD45 and CD34 antibodies. The number of isolated mMSCs as estimated by colony forming unit-fibroblast (CFU-F) assay showed that this modified isolation method could yield 70.0% more primary colonies. After immunodepletion, a homogenous mMSC population could already be obtained after two passages. Immunodepleted mMSCs (ID-mMSCs) are uniformly positive for stem cell antigen-1 (Sca-1), CD90, CD105 and CD73 cell surface markers, but negative for the hematopoietic surface markers CD14, CD34 and CD45. Moreover the immunodepleted cell population exhibits more differentiation potential into adipogenic, osteogenic and chondrogenic lineages. Our data illustrate the development of an efficient and reliable expansion protocol increasing the yield and purity of mMSCs and reducing the overall expansion time.

scholarly journals ID:2050 Impacts of polymer-based and graphene-based biomaterials on the characteristics of Wharton jelly’s-derived mesenchymal stem cells

2017 ◽  
Vol 4 (S) ◽  
pp. 80
Author(s):  
Hiew Vun Vun
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Molecular Mechanisms ◽  
Surface Markers ◽  
Rt Pcr ◽  
Alizarin Red ◽  
Runx2 Expression ◽  
Gene Profiles ◽  
Different Types ◽  
Different Characteristics

The combination of mesenchymal stem cells (MSCs) and biomaterials scaffolds hold significant promise in tissue engineering and regenerative medicine field. In recent years, expansion and differentiation of MSCs culturing with polymer-based and graphene-based biomaterials have been intensely studied. However, the underlying molecular mechanisms are still largely unknown. This study was to examine the different characteristics of Wharton’s jelly (WJ)-derived MSCs using two different types of biomaterials including polymer-based (PU157) and graphene oxide-based (GO). In term of proliferation, WJ-MSCs culturing with PU157 and GO showed no significant differences compared to the controls. PU157 and GO had no effects on morphological features of WJ-MSCs. RT-PCR results showed that all WJ-MSCs co-culturing with or without PU157 and GO expressed positive surface markers including CD29, CD44, CD73, CD90, CD105, CD106 and CD166 but the expression of negative markers (CD34, CD45 and CD133) was undetectable. The SOX2 and RUNX2 expression were both upregulated in WJ-MSCs combining with PU157 when compared to the controls at passage 6, where no obvious difference was observed in passage 3. Interestingly, COL2A expression became more noticeable in the presence of PU157 at passage 6 than passage 3. In contrast, GO did not alter the expression of stemness, osteogenic, adipogenic and chondrogenic genes. Both alizarin red and oil red staining showed calcium and lipid deposition in WJ-MSCs co-cultured with PU157 and GO respectively, suggesting the presence of osteoblast- and adipocyte-like cells. In conclusion, PU157 exerted certain effects on the expression gene profiles at later passage and able to enhance differentiation abilities in WJ-MSCs.

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.

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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