279 COMPARISONS OF BONE MARROW AND PUTATIVE SKIN-DERIVED MESENCHYMAL STEM CELLS AS DONOR FOR NUCLEAR TRANSFER IN MINIATURE PIG

2009 ◽  
Vol 21 (1) ◽  
pp. 236
Author(s):  
E. J. Kang ◽  
B. U. Park ◽  
H. J. Song ◽  
Y. I. Yang ◽  
M. J. Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Mrna Expression ◽  
Nuclear Transfer ◽  
Miniature Pig ◽  
Rt Pcr ◽  
Donor Cells ◽  
Lipid Vacuoles ◽  
Histological Staining

Since the birth of the first cloned sheep was reported, fibroblasts are commonly used as donor cells for nuclear transfer. However, in some species there is a higher incidence of abnormal fetuses, still births, and neonatal deaths as a consequence of reprogramming disorders including DNA methylation and histone acetylation. Cloned embryos with mesenchymal stem cells (MSC) have shown higher developmental ability compared to fibroblast, suggesting that undifferentiated genome can required ease reprogramming. Because MSC are relatively difficult to collect from bone marrow, skin is an alternative source of the donor cells. However, molecular and functional analyses remain uncertain between MSC derived from bone marrow and skin stem cells isolated from ear. The present study compared the expression of early transcription factors (Oct-4, Nanog and Sox-2), and differentiation capability to osteocytes, adipocytes and chondrocytes of MSC isolated from bone marrow and skin-derived putative stem cells from miniature pig. Bone marrow was isolated by Ficoll density gradient method, and skin separated from epidermis and dermis was diced into 2-mm diameter explants, and attached to tissue culture dishes. Cells were then cultured in DMEM/F12 supplemented with 10% FBS, 10 ng mL–1 bFGF, 10 ng mL–1 EGF at 38.5°C, in a humidified atmosphere of 5% CO2 in air. Expression of Oct-4, Nanog, Sox2 was analysed by RT-PCR. Osteogenic and adipogenic differentiation were induced following the protocols described previously (Jin et al. 2007 Int. J. Dev. Biol. 51, 85–90; Mohana Kumar et al. 2007 Mol. Cells 24, 343–350) and compared by histological staining and RT-PCR. Osteocytes were defined by the formation of the mineral nodules of deposition of calcium by Von Kossa staining and differentiations into adipocytes and chondrocytes were identified by oil red O staining of lipid vacuoles and alcian blue of proteoglycan, respectively. Skin-derived MSC were revealed to similar mRNA expression of Oct-4, Nanog, Sox2 compared to bone marrow derived MSC. However, bone marrow derived MSC were higher mRNA expression about of osteocytic genes (osteoclacin and osteonectin), chondrocytic gene (collagen type) and adipocytic genes (aP2) than those of skin-derived MSC. In addition, bone marrow derived MSC were revealed greater deposition of calcium, proteoglycan, and lipid vacuoles than those of skin derived MSC by histological staining. The results of present study suggest that cells isolated from skin have lower potential than MSC isolated from bone marrow. However, skin-derived stem cells have properties of multi-lineage differentiation and can be obtained easily. These stem cells, therefore, can serve as easily accessible and expandable source possessing donor cells for cloning, potential animal model, and clinical applications. This work was supported by Grant No. 20070301034040 from Bio-organ, Republic of Korea.

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

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

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

43 PRODUCTION OF PORCINE EMBRYOS BY NUCLEAR TRANSFER OF BONE MARROW MESENCHYMAL STEM CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 130
Author(s):  
H.-F. Jin ◽  
B. Mohana Kumar ◽  
J.-G. Kim ◽  
H.-J. Song ◽  
S. Balasubramanian ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Nuclear Transfer ◽  
Total Cell ◽  
Differentiation Potential ◽  
Developmental Potential ◽  
Cell Numbers ◽  
Donor Cells ◽  
Fetal Fibroblasts

Recent experimental evidence indicates that adult stem cells are more desirable than somatic cells for nuclear transfer (NT) because of their easy reprogrammability to resemble the genome of the zygote (Zhu et al. 2004 Biol. Reprod. 71, 1890-1897). Mesenchymal stem cells (MSCs) are a heterogeneous population of uncommitted and lineage-committed cells and may have a more flexible potential as donor cells for NT. The aim of this study was to characterize an isolated population of porcine MSCs from bone marrow and to compare the developmental potential of cloned (IVF) embryos with MSCs and porcine fetal fibroblasts (pFFs) by assessing the cleavage and blastocyst rate, total cell numbers, inner cell mass (ICM) ratio and apoptosis. MSCs were obtained from the aspirated bone marrow of 6-8-month-old pigs. Cells were centrifuged, resuspended, and plated in advanced-DMEM (ADMEM) supplemented with 5% fetal bovine serum (FBS). The differentiation potential was demonstrated by culture of MSCs (passage 3) under conditions that were favorable for adipogenic, osteogenic, and chondrogenic development (Pittenger et al. 1999 Science 284, 143-147). Oil red O staining revealed that MSCs produced lipid droplets after incubation in adipogenic media. Following osteoinduction, MSCs exhibited robust alkaline phosphatase activity and cells later transformed into mineralized nodules as demonstrated by von Kossa staining. Histological staining of proteoglycan indicated chondrogenic differentiation. Cumulus-oocyte complexes were matured, fertilized, and cultured by the following method (Abeydeera et al. 2000 Theriogenology 54, 787-797). NT embryos were produced as described by Kim et al. (2005 Mol. Rep. Dev. 70, 308-313). Cleavage rate was significantly (P < 0.05) higher in IVF embryos than in NT embryos derived from MSCs and pFFs (84.5% � 4.6 vs. 52.2% � 5.4 and 50.8% � 5.2, respectively). However, blastocyst rates in IVF embryos and NT embryos derived from MSCs (20.6% � 2.5 and 18.5% � 3.0) did not differ but these rates were significantly (P < 0.05) higher than that for NT embryos derived from pFFs (9.5% � 2.1). Total cell numbers and the ratio of ICM to total cells among embryos developed in NT from MSCs (29.4 � 5.2 and 0.38 � 0.08, respectively) were significantly (P < 0.05) higher than for those from pFFs (22.6 � 5.5 and 0.18 � 0.12, respectively). Proportions of TUNEL-positive cells in NT embryos from pFFs (12.8 � 2.5) were significantly (P < 0.05) higher than in those from MSCs (8.6 � 1.8) and in IVF embryos (4.6 � 1.5). The results clearly demonstrate that multipotent bone marrow MSCs can make a suitable alternative to fibroblasts as donor cells and have a greater potential for producing viable cloned porcine embryos. This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.

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.

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.

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.

Notch Signaling Pathway in Hematopoietic Stem Cells Is Activated through Interactive Communication with Mesenchymal Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1408-1408
Author(s):  
Yuji Kikuchi ◽  
Akihiro Kume ◽  
Masashi Urabe ◽  
Hiroaki Mizukami ◽  
Takahiro Suzuki ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Notch Signaling ◽  
Notch Signaling Pathway ◽  
Cell Contact ◽  
Rt Pcr ◽  
Interactive Communication ◽  
Hematopoietic Stem ◽  
Notch Ligands

Abstract Mesenchymal stem cells (MSCs), which are key elements of hematopoietic microenvironment in bone marrow, are known to play a critical role in supporting hematopoiesis. A variety of hematopoietic growth factors are produced from MSCs, and cell-to-cell contact is also believed to be crucial in the interaction between hematopoietic stem cells (HSCs) and MSCs. However, the molecular mechanisms of hematopoiesis-supporting ability of MSCs are still unclear. In particular, there is little information regarding the effects of HSCs on MSC function. In the present study, we investigated the cellular and molecular events in the interactive communication between HSCs and MSCs using a differentiation-inducible MSC model; i.e. parent C3H10T1/2 cells and 10T1/2-derived cell lines, A54 preadipocytes and M1601 myoblasts. These cells were co-cultured with murine HSCs (Lin-Sca1+) isolated from bone marrow. There was 9-fold increase in the number of hematopoietic progenitors after co-culture with A54 preadipocytes, whereas there was no increase when co-cultured with parent 10T1/2 or M1601 cells. More intriguingly, cobblestone areas were observed only when HSCs were co-cultured with A54 cells. Quantitative RT-PCR showed that A54 cells express significantly higher levels of SCF, SDF-1, and angiopoietin-1 (Ang-1) compared with parent 10T1/2 cells and M1601 cells, although these cytokines were not up-regulated when co-cultured with HSCs. To search for the genes involved in the interaction between HSCs and MSCs, we compared gene expression profiles before and after the co-culture by using a microarray analysis. Among the candidate genes with up-regulation after the co-culture, we paid attention to the Notch system, because Notch ligands are considered to play an important role in nurturing HSCs within the hematopoietic microenvironment. As a result, the expression of Notch ligands, Jagged1 and Dll3, increased in A54 cells after the coculture with HSCs. On the other hand, the expression of Notch1 and Hes-1 also increased in HSCs upon co-culture with A54 cells. These data were confirmed by quantitative RT-PCR. Moreover, when HSCs were co-cultured with A54 cells without cell-to-cell contact using Transwell permeable supports, there was neither increase in the number of progenitors in the upper wells, nor the up-regulation of Notch ligands in A54 cells in the lower wells. These findings support the idea that HSCs act on MSCs to induce the expression of Notch ligands via direct cell-to-cell contact and that the Notch ligands derived from MSCs act on HSCs in turn to activate Notch signaling pathway, possibly leading to the cobblestone formation with the maintenance of immature state of HSCs. The Notch system may be one of the critical elements in the interactive communication between HSCs and MSCs.

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.

Functional ion channels in mouse bone marrow mesenchymal stem cells

2007 ◽  
Vol 293 (5) ◽  
pp. C1561-C1567 ◽  
Author(s):  
Rong Tao ◽  
Chu-Pak Lau ◽  
Hung-Fat Tse ◽  
Gui-Rong Li
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Ion Channel ◽  
Mouse Bone Marrow ◽  
Bone Marrow Mscs ◽  
Rt Pcr ◽  
Western Immunoblotting ◽  
Immunoblotting Analysis ◽  
Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (MSCs) are used as a cell source for cardiomyoplasty; however, the cellular electrophysiological properties are not fully understood. The present study was to investigate the functional ionic channels in undifferentiated mouse bone marrow MSCs using whole cell patch-voltage clamp technique, RT-PCR, and Western immunoblotting analysis. We found that three types of ionic currents were present in mouse MSCs, including a Ca2+-activated K+ current ( IKCa), an inwardly rectifying K+ current ( IKir), and a chloride current ( ICl). IKir was inhibited by Ba2+, and IKCa was activated by the Ca2+ ionophore A-23187 and inhibited by the intermediate-conductance IKCa channel blocker clotrimazole. ICl was activated by hyposmotic (0.8 T) conditions and inhibited by the chloride channel blockers DIDS and NPPB. The corresponding ion channel genes and proteins, KCa3.1 for IKCa, Kir2.1 for IKir, and Clcn3 for ICl, were confirmed by RT-PCR and Western immunoblotting analysis in mouse MSCs. These results demonstrate that three types of functional ion channel currents (i.e., IKir, IKCa, and ICl) are present in mouse bone marrow MSCs.

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