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.
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.
Canine mesenchymal stem cells (cMSC) have been successfully isolated from several adult tissue sources. However, differences in the biological properties of MSC have been shown to be associated with donor variability. Further, the stem cell capacity of cMSC of various tissues isolated from a single donor is currently unclear. Therefore, this study investigated the functional and molecular characteristics of cMSC derived from bone marrow (cBM-MSC), adipose tissue (cA-MSC) and dermal skin (cDS-MSC) of a single donor. Three kinds of cMSC were isolated by following previously published protocols. AP activity was assessed with a chromogen kit (Abcam Inc., Cambridge, MA, USA). Expression of CD markers (CD45, 90 and 105) and stem cell transcription factors (Oct3/4, Nanog and Sox2) was analysed by immunocytochemical staining. All cells were induced into osteogenesis and adipogenesis by following protocols described earlier and confirmed by cytochemical staining and the detection of lineage specific genes by RT-PCR. Chromosomal stability was assessed by a method described earlier (Ock and Rho 2008 J. Vet. Med. Sci. 70, 1165–1172) and cell cycle status was determined by a flow cytometry. Telomere length was analysed by Telo TAGGG Telomere Length Assay kit (Roche, Mannheim, Germany) and telomerase activity was evaluated by semiquantitative nested RT-PCR. Statistical analysis was performed by ANOVA using SPSS 12.0 and significance was tested when P < 0.05. Expressions of AP activity and the transcription factors, such as Oct3/4, Nanog and Sox2 were absent in all cMSC. All 3 types of cMSC positively expressed the surface markers CD90 and 105 but not CD45. Exposure of all cell lines to osteogenic and adipogenic induction medium resulted in the calcium deposition evidenced by Alizarin red S staining and the accumulation of fat globules indicated by Oil red O staining, respectively. Differentiation was further confirmed by the detection of marker genes, such as Runx2 and Pparγ. However, the degree of osteogenic or adipogenic differentiation among the 3 kinds of cMSC was different and particularly, cA-MSC had enhanced cytochemical staining associated with expression of specific genes, Runx2 and Pparγ. Ploidy analysis showed that the diploid rate was high with over 90% in all cMSC and indicated no noticeable chromosomal abnormalities. Further, less than 52% of cells were found at G1 phase in all cMSC, with lowest percentage observed in cDS-MSC (33.3%). Regardless of varied tissue sources, cMSC from a single donor showed no differences in telomere lengths (∼18–19 kbp), but the telomerase activity was different with significantly higher levels found in cBM-MSC. In conclusion, the above results suggest that tissue specific cMSC derived from a single donor possess differences in stem cell capacity and support the consideration of tissue source before judging the suitability of cells for therapeutic applications. This work was supported by grant from Basic Science Research Program through NRF funded by the Ministry of Education, Science and Technology (2009-0064229).
In the context of multipotent stem cells, mesenchymal stem cells (MSC) derived from bone marrow have been identified as most promising cell types for the treatment of smooth muscle related injured tissues and organs. In the present study, the ability of porcine bone marrow derived MSC to differentiate in vitro into smooth muscle cells (SMC) was examined. MSC were isolated from domestic pig bone marrow by their readily adherent property to tissue culture plastic with fibroblast-like morphology. Cells were analysed for the expression of MSC specific markers by flow cytometer and mesenchymal lineage differentiation by following previously published protocols. Differences in values were analysed by one-way ANOVA using SPSS and data are presented as mean ± SD. Flow cytometry analysis of MSC showed the positive expression of markers, such as CD29 (97.33 ± 2.08%), CD44 (97.67 ± 1.15%), CD73 (62.33 ± 2.89%), CD90 (96.67 ± 2.08%) and vimentin (59.33 ± 2.52%). In contrast, the expression levels were significantly lower for CD34 (3.33 ± 1.53%), CD45 (3.67 ± 1.53%), major histocompatibility complex class II (MHC class II, 10.33 ± 2.52%) and swine leukocyte antigen-DR (SLA-DR, 9.67 ± 2.08%). The MSC were further confirmed by their ability to differentiate in vitro along the distinct lineages of adipocytes (Oil red O), osteocytes (von Kossa and Alizarin red) and chondrocytes (Alcian blue). Induction of SMC differentiation was performed with supplementation of porcine transforming growth factor-β (TGF-β) and recombinant human bone morphogenic protein 4 (BMP4) as described earlier (Wang et al. 2010 Tissue Eng. A 1201–1213) with minor modifications. Upon induction, porcine MSC acquired myoblast-like morphology with intracellular thin filaments. Immunofluorescence staining showed the presence of early and late markers of smooth muscle differentiation, such as α-smooth muscle actin (α-SMA), calponin, smooth muscle 22 α (SM22α) and smooth muscle-myosin heavy chain (SM-MHC) and their expression levels varied from 22.65% to 56.75%. Later, the expression of selected markers was demonstrated by Western blotting analysis. Consistent with this phenotypic characterisation, reverse transcription-polymerase chain reaction (RT-PCR) and quantitative PCR (RT-qPCR) further showed the expression and a sequential up-regulation of transcripts for α-SMA, calponin, SM22α and SM-MHC. However, no expression of SMC-specific markers was observed in untreated MSC. In conclusion, these findings suggest the ability of porcine MSC from bone marrow to differentiate in vitro into SMC in the presence of growth factors. Further understanding of SMC differentiation with functional properties would be essential for employing porcine MSC as a useful model for cell-based tissue engineering and regeneration strategies. 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.
Generation of induced pluripotent stem cells (iPSC) present not only an opportunity to use the autologous cells clinically but also as cellular models for studying molecular mechanisms of various diseases and screening of drugs. The objective of the present study was to generate iPSC-like cells from rat ear skin cells by evaluating the expression of alkaline phosphatase (AP) activity, detection of markers specific to embryonic stem cells (ESC) and in vitro differentiation into selected cell lineages. Ear skin cells were isolated from 6-week-old s.d. rats and cultured in advanced-DMEM supplemented with 10% FBS at 37°C in a humidified atmosphere of 5% CO2 in air. Cells were reprogrammed with retroviral factors, namely OCT4, SOX2, KLF4 and c-Myc, the combination commonly used to generate mouse and human iPSCs. These cells were cultured in media specific to ESC on mitomycin treated mouse embryonic fibroblasts at 37°C in a humidified atmosphere of 5% CO2 in air. AP activity was assessed with Western Blue® kit (Promega, Madison, WI, USA). Expressions of ESC specific markers, such as OCT4, NANOG, SSEA-1, SSEA-4 and REX1 were evaluated by reverse transcriptase-PCR (RT-PCR). Reprogrammed iPSC-like cells were differentiated into adipogenic, osteogenic and neurogenic lineages by following previously described protocols. Cytochemical staining was performed to confirm the adipogenesis and osteogenesis. Further, the lineage specific marker genes in adipocytes (PPARγ2, adiponectin and aP2), osteocytes (osteocalcin, osteonectin, osteopontin and Runx2) and neuronal cells (nestin, neurogenin-1, β-tubulin and nerve growth factor) were evaluated by RT-PCR. Following transduction, ear skin cells were successfully reprogrammed into iPSC-like cells. Generated iPSC-like cells were positive for AP activity and clearly expressed the markers of ESC, including OCT4, NANOG, SSEA-1, SSEA-4 and REX1. After induction, iPSC-like cells were capable of differentiating in vitro into adipocytes as indicated by Oil red O staining and expressed the specific markers, such as PPARγ2, adiponectin and aP2. Differentiation of iPSC-like cells into osteocytes was evidenced by von Kossa staining and the detection of marker genes, osteocalcin, osteonectin, osteopontin and Runx2. Furthermore, upon neuronal specific induction, iPSC-like cells were able to form neuronal cells as demonstrated by the expression of neuronal specific markers, such as nestin, neurogenin-1, β-tubulin and nerve growth factor. In conclusion, our results showed the successful generation of iPSC-like cells from rat ear skin cells by exhibiting similar features of ESCs and demonstrated their ability to differentiate in vitro into adipocytes, osteocytes and neuronal cells. Further studies are being carried out to characterise and demonstrate the pluripotency capacity of generated iPS-like cells in a rat model. This work was supported by Grant No. 2007031034040 from Bio-organ and Grant No. 200908FHT010204005 from Biogreen21.
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.
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.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.