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1.  Hepatogenic and neurogenic differentiation of bone marrow mesenchymal stem cells from abattoir-derived bovine fetuses 
BMC Veterinary Research  2014;10:154.
Background
Mesenchymal stem cells (MSC) are multipotent progenitor cells characterized by their ability to both self-renew and differentiate into tissues of mesodermal origin. The plasticity or transdifferentiation potential of MSC is not limited to mesodermal derivatives, since under appropriate cell culture conditions and stimulation by bioactive factors, MSC have also been differentiated into endodermal (hepatocytes) and neuroectodermal (neurons) cells. The potential of MSC for hepatogenic and neurogenic differentiation has been well documented in different animal models; however, few reports are currently available on large animal models. In the present study we sought to characterize the hepatogenic and neurogenic differentiation and multipotent potential of bovine MSC (bMSC) isolated from bone marrow (BM) of abattoir-derived fetuses.
Results
Plastic-adherent bMSC isolated from fetal BM maintained a fibroblast-like morphology under monolayer culture conditions. Flow cytometric analysis demonstrated that bMSC populations were positive for MSC markers CD29 and CD73 and pluripotency markers OCT4 and NANOG; whereas, were negative for hematopoietic markers CD34 and CD45. Levels of mRNA of hepatic genes α-fetoprotein (AFP), albumin (ALB), alpha1 antitrypsin (α1AT), connexin 32 (CNX32), tyrosine aminotransferase (TAT) and cytochrome P450 (CYP3A4) were up-regulated in bMSC during a 28-Day period of hepatogenic differentiation. Functional analyses in differentiated bMSC cultures evidenced an increase (P < 0.05) in albumin and urea production and glycogen storage. bMSC cultured under neurogenic conditions expressed NESTIN and MAP2 proteins at 24 h of culture; whereas, at 144 h also expressed TRKA and PrPC. Levels of MAP2 and TRKA mRNA were up-regulated at the end of the differentiation period. Conversely, bMSC expressed lower levels of NANOG mRNA during both hepatogenic and neurogenic differentiation processes.
Conclusion
The expression patterns of linage-specific markers and the production of functional metabolites support the potential for hepatogenic and neurogenic differentiation of bMSC isolated from BM of abattoir-derived fetuses. The simplicity of isolation and the potential to differentiate into a wide variety of cell lineages lays the foundation for bMSC as an interesting alternative for investigation in MSC biology and eventual applications for regenerative therapy in veterinary medicine.
doi:10.1186/1746-6148-10-154
PMCID: PMC4098697  PMID: 25011474
Bovine fetuses; Mesenchymal stem cell; Differentiation potential; Hepatocyte-like cell; Neuron-like cell
2.  Isolation and multilineage differentiation of bone marrow mesenchymal stem cells from abattoir-derived bovine fetuses 
Background
Mesenchymal stem cells (MSC) are multipotent progenitor cells localized in the stromal compartment of the bone marrow (BM). The potential of MSC for mesenchymal differentiation has been well documented in different animal models predominantly on rodents. However, information regarding bovine MSC (bMSC) is limited, and the differentiation potential of bMSC derived from fetal BM remains unknown. In the present study we sought to isolate bMSC from abattoir-derived fetal BM and to characterize the multipotent and differentiation potential under osteogenic, chondrogenic and adipogenic conditions by quantitative and qualitative analyses.
Results
Plastic-adherent bMSC isolated from fetal BM maintained a fibroblast-like morphology under monolayer culture conditions. These cells expressed high levels of MSC surface markers (CD73, CD90, and CD105) and low levels of hematopoietic surface markers (CD34 and CD45). Culture of bMSC under osteogenic conditions during a 27-day period induced up-regulation of the osteocalcin (OC) gene expression and alkaline phosphatase (ALPL) activity, and promoted mineralization of the matrix. Increasing supplementation levels of ascorbic acid to culture media enhanced osteogenic differentiation of bMSC; whereas, reduction of FBS supplementation compromised osteogenesis. bMSC increased expression of cartilage-specific genes aggrecan (ACAN), collagen 2A1 (COL2A1) and SRY (sex-determining region Y) box 9 (SOX9) at Day 21 of chondrogenic differentiation. Treatment of bMSC with adipogenic factors increased levels of fatty acid-binding protein 2 (AP2) mRNA and accumulation of lipid vacuoles after 18 days of culture. NANOG mRNA levels in differentiating bMSC were not affected during adipogenic culture; however, osteogenic and chondrogenic conditions induced higher and lower levels, respectively.
Conclusions
Our analyses revealed the potential multilineage differentiation of bMSC isolated from abattoir-derived fetal BM. NANOG mRNA pattern in differentiating bMSC varied according to differentiation culture conditions. The osteogenic differentiation of bMSC was affected by ascorbic acid and FBS concentrations in culture media. The simplicity of isolation and the differentiation potential suggest that bMSC from abattoir-derived fetal BM are appropriate candidate for investigating MSC biology and for eventual applications for regenerative therapy.
doi:10.1186/1746-6148-9-133
PMCID: PMC3751243  PMID: 23826829
Mesenchymal stem cell; Bovine fetuses; Differentiation potential; Multipotency
3.  Equine mesenchymal stem cells from bone marrow, adipose tissue and umbilical cord: immunophenotypic characterization and differentiation potential 
Introduction
Studies with mesenchymal stem cells (MSCs) are increasing due to their immunomodulatory, anti-inflammatory and tissue regenerative properties. However, there is still no agreement about the best source of equine MSCs for a bank for allogeneic therapy. The aim of this study was to evaluate the cell culture and immunophenotypic characteristics and differentiation potential of equine MSCs from bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and umbilical cord (UC-MSCs) under identical in vitro conditions, to compare these sources for research or an allogeneic therapy cell bank.
Methods
The BM-MSCs, AT-MSCs and UC-MSCs were cultured and evaluated in vitro for their osteogenic, adipogenic and chondrogenic differentiation potential. Additionally, MSCs were assessed for CD105, CD44, CD34, CD90 and MHC-II markers by flow cytometry, and MHC-II was also assessed by immunocytochemistry. To interpret the flow cytometry results, statistical analysis was performed using ANOVA.
Results
The harvesting and culturing procedures of BM-MSCs, AT-MSCs and UC-MSCs were feasible, with an average cell growth until the third passage of 25 days for BM-MSCs, 15 days for AT-MSCs and 26 days for UC-MSCs. MSCs from all sources were able to differentiate into osteogenic (after 10 days for BM-MSCs and AT-MSCs and 15 days for UC-MSCs), adipogenic (after 8 days for BM-MSCs and AT-MSCs and 15 days for UC-MSCs) and chondrogenic (after 21 days for BM-MSCs, AT-MSCs and UC-MSCs) lineages. MSCs showed high expression of CD105, CD44 and CD90 and low or negative expression of CD34 and MHC-II. The MHC-II was not detected by immunocytochemistry techniques in any of the MSCs studied.
Conclusions
The BM, AT and UC are feasible sources for harvesting equine MSCs, and their immunophenotypic and multipotency characteristics attained minimal criteria for defining MSCs. Due to the low expression of MHC-II by MSCs, all of the sources could be used in clinical trials involving allogeneic therapy in horses. However, the BM-MSCs and AT-MSCs showed fastest ‘‘in vitro’’ differentiation and AT-MSCs showed highest cell growth until third passage. These findings suggest that BM and AT may be preferable for cell banking purposes.
doi:10.1186/scrt414
PMCID: PMC4055040  PMID: 24559797
4.  Sox2 Suppression by miR-21 Governs Human Mesenchymal Stem Cell Properties 
The miRNA profile of mesenchymal stem cells (MSCs) derived from amniotic fluid, bone marrow (BM), and umbilical cord blood was analyzed. Initially, 67 different miRNAs were identified that were expressed in all three types of MSCs but at different levels, depending on the source. A more detailed analysis revealed that miR-21 was expressed at higher levels in RS-AF-MSCs and BM-MSCs compared with SS-AF-MSCs. Findings suggest that miR-21 might function by regulating Sox2 expression in human MSCs and might also act as a key molecule determining MSC proliferation and differentiation.
MicroRNAs (miRNAs) have recently been shown to act as regulatory signals for maintaining stemness and for determining the fate of adult and fetal stem cells, such as human mesenchymal stem cells (hMSCs). hMSCs constitute a population of multipotent stem cells that can be expanded easily in culture and are able to differentiate into many lineages. We have isolated two subpopulations of fetal mesenchymal stem cells (MSCs) from amniotic fluid (AF) known as spindle-shaped (SS) and round-shaped (RS) cells and characterized them on the basis of their phenotypes, pluripotency, proliferation rates, and differentiation potentials. In this study, we analyzed the miRNA profile of MSCs derived from AF, bone marrow (BM), and umbilical cord blood (UCB). We initially identified 67 different miRNAs that were expressed in all three types of MSCs but at different levels, depending on the source. A more detailed analysis revealed that miR-21 was expressed at higher levels in RS-AF-MSCs and BM-MSCs compared with SS-AF-MSCs. We further demonstrated for the first time a direct interaction between miR-21 and the pluripotency marker Sox2. The induction of miR-21 strongly inhibited Sox2 expression in SS-AF-MSCs, resulting in reduced clonogenic and proliferative potential and cell cycle arrest. Strikingly, the opposite effect was observed upon miR-21 inhibition in RS-AF-MSCs and BM-MSCs, which led to an enhanced proliferation rate. Finally, miR-21 induction accelerated osteogenesis and impaired adipogenesis and chondrogenesis in SS-AF-MSCs. Therefore, these findings suggest that miR-21 might specifically function by regulating Sox2 expression in human MSCs and might also act as a key molecule determining MSC proliferation and differentiation.
doi:10.5966/sctm.2013-0081
PMCID: PMC3902287  PMID: 24307698
Amniotic fluid mesenchymal stem cells; Bone marrow mesenchymal stem cells; Umbilical cord blood mesenchymal stem cells; miR-21; Sox2; Cell cycle
5.  Effect of hypoxia on equine mesenchymal stem cells derived from bone marrow and adipose tissue 
Background
Mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) are being applied to equine cell therapy. The physiological environment in which MSCs reside is hypoxic and does not resemble the oxygen level typically used in in vitro culture (20% O2). This work compares the growth kinetics, viability, cell cycle, phenotype and expression of pluripotency markers in both equine BM-MSCs and AT-MSCs at 5% and 20% O2.
Results
At the conclusion of culture, fewer BM-MSCs were obtained in hypoxia than in normoxia as a result of significantly reduced cell division. Hypoxic AT-MSCs proliferated less than normoxic AT-MSCs because of a significantly higher presence of non-viable cells during culture. Flow cytometry analysis revealed that the immunophenotype of both MSCs was maintained in both oxygen conditions. Gene expression analysis using RT-qPCR showed that statistically significant differences were only found for CD49d in BM-MSCs and CD44 in AT-MSCs. Similar gene expression patterns were observed at both 5% and 20% O2 for the remaining surface markers. Equine MSCs expressed the embryonic markers NANOG, OCT4 and SOX2 in both oxygen conditions. Additionally, hypoxic cells tended to display higher expression, which might indicate that hypoxia retains equine MSCs in an undifferentiated state.
Conclusions
Hypoxia attenuates the proliferative capacity of equine MSCs, but does not affect the phenotype and seems to keep them more undifferentiated than normoxic MSCs.
doi:10.1186/1746-6148-8-142
PMCID: PMC3483288  PMID: 22913590
Hypoxia; Horse; AT-MSC; BM-MSC; Characterisation
6.  Phenotype, donor age and gender affect function of human bone marrow-derived mesenchymal stromal cells 
BMC Medicine  2013;11:146.
Background
Mesenchymal stromal cells (MSCs) are attractive for cell-based therapies ranging from regenerative medicine and tissue engineering to immunomodulation. However, clinical efficacy is variable and it is unclear how the phenotypes defining bone marrow (BM)-derived MSCs as well as donor characteristics affect their functional properties.
Methods
BM-MSCs were isolated from 53 (25 female, 28 male; age: 13 to 80 years) donors and analyzed by: (1) phenotype using flow cytometry and cell size measurement; (2) in vitro growth kinetics using population doubling time; (3) colony formation capacity and telomerase activity; and (4) function by in vitro differentiation capacity, suppression of T cell proliferation, cytokines and trophic factors secretion, and hormone and growth factor receptor expression. Additionally, expression of Oct4, Nanog, Prdm14 and SOX2 mRNA was compared to pluripotent stem cells.
Results
BM-MSCs from younger donors showed increased expression of MCAM, VCAM-1, ALCAM, PDGFRβ, PDL-1, Thy1 and CD71, and led to lower IL-6 production when co-cultured with activated T cells. Female BM-MSCs showed increased expression of IFN-γR1 and IL-6β, and were more potent in T cell proliferation suppression. High-clonogenic BM-MSCs were smaller, divided more rapidly and were more frequent in BM-MSC preparations from younger female donors. CD10, β1integrin, HCAM, CD71, VCAM-1, IFN-γR1, MCAM, ALCAM, LNGFR and HLA ABC were correlated to BM-MSC preparations with high clonogenic potential and expression of IFN-γR1, MCAM and HLA ABC was associated with rapid growth of BM-MSCs. The mesodermal differentiation capacity of BM-MSCs was unaffected by donor age or gender but was affected by phenotype (CD10, IFN-γR1, GD2). BM-MSCs from female and male donors expressed androgen receptor and FGFR3, and secreted VEGF-A, HGF, LIF, Angiopoietin-1, basic fibroblast growth factor (bFGF) and NGFB. HGF secretion correlated negatively to the expression of CD71, CD140b and Galectin 1. The expression of Oct4, Nanog and Prdm14 mRNA in BM-MSCs was much lower compared to pluripotent stem cells and was not related to donor age or gender. Prdm14 mRNA expression correlated positively to the clonogenic potential of BM-MSCs.
Conclusions
By identifying donor-related effects and assigning phenotypes of BM-MSC preparations to functional properties, we provide useful tools for assay development and production for clinical applications of BM-MSC preparations.
doi:10.1186/1741-7015-11-146
PMCID: PMC3694028  PMID: 23758701
Mesenchymal stromal/stem cells; Age; Gender; Immunomodulation; Phenotype; Differentiation
7.  Fetal cardiac mesenchymal stem cells express embryonal markers and exhibit differentiation into cells of all three germ layers 
World Journal of Stem Cells  2013;5(1):26-33.
AIM: To study the expression of embryonal markers by fetal cardiac mesenchymal stem cells (fC-MSC) and their differentiation into cells of all the germ layers.
METHODS: Ten independent cultures of rat fC-MSC were set up from cells derived from individual or pooled fetal hearts and studies given below were carried out at passages 3, 6, 15 and 21. The phenotypic markers CD29, CD31, CD34, CD45, CD73, CD90, CD105, CD166 and HLA-DR were analyzed by flow cytometry. The expression of embryonal markers Oct-4, Nanog, Sox-2, SSEA-1, SSEA-3, SSEA-4, TRA-1-60 and TRA 1-81 were studied by immunocytochemistry. The fC-MSC treated with specific induction medium were evaluated for their differentiation into (1) adipocytes and osteocytes (mesodermal cells) by Oil Red O and Alizarin Red staining, respectively, as well as by expression of lipoprotein lipase, PPARγ2 genes in adipocytes and osteopontin and RUNX2 genes in osteocytes by reverse-transcription polymerase chain reaction (RT-PCR); (2) neuronal (ectodermal) cells by expression of neuronal Filament-160 and Glial Fibrillar Acidic Protein by RT-PCR and immunocytochemistry; and (3) hepatocytic (endodermal) cells by expression of albumin by RT-PCR and immunocytochemistry, glycogen deposits by Periodic Acid Schiff staining and excretion of urea into the culture supernatant.
RESULTS: The fC-MSC expressed CD29, CD73, CD90, CD105, CD166 but lacked expression of CD31, CD34, CD45 and HLA-DR. They expressed embryonal markers, viz. Oct-4, Nanog, Sox-2, SSEA-1, SSEA-3, SSEA-4, TRA-1-81 but not TRA-1-60. On treatment with specific induction media, they differentiated into adipocytes and osteocytes, neuronal cells and hepatocytic cells.
CONCLUSION: Our results together suggest that fC-MSC are primitive stem cell types with a high degree of plasticity and, in addition to their suitability for cardiovascular regenerative therapy, they may have a wide spectrum of therapeutic applications in regenerative medicine.
doi:10.4252/wjsc.v5.i1.26
PMCID: PMC3557348  PMID: 23362437
Fetal cardiac mesenchymal stem cells; Embryonal markers; Multipotent differentiation potential
8.  Decreased hypertrophic differentiation accompanies enhanced matrix formation in co-cultures of outer meniscus cells with bone marrow mesenchymal stromal cells 
Arthritis Research & Therapy  2012;14(3):R153.
Introduction
The main objective of this study was to determine whether meniscus cells from the outer (MCO) and inner (MCI) regions of the meniscus interact similarly to or differently with mesenchymal stromal stem cells (MSCs). Previous study had shown that co-culture of meniscus cells with bone marrow-derived MSCs result in enhanced matrix formation relative to mono-cultures of meniscus cells and MSCs. However, the study did not examine if cells from the different regions of the meniscus interacted similarly to or differently with MSCs.
Methods
Human menisci were harvested from four patients undergoing total knee replacements. Tissue from the outer and inner regions represented pieces taken from one third and two thirds of the radial distance of the meniscus, respectively. Meniscus cells were released from the menisci after collagenase treatment. Bone marrow MSCs were obtained from the iliac crest of two patients after plastic adherence and in vitro culture until passage 2. Primary meniscus cells from the outer (MCO) or inner (MCI) regions of the meniscus were co-cultured with MSCs in three-dimensional (3D) pellet cultures at 1:3 ratio, respectively, for 3 weeks in the presence of serum-free chondrogenic medium containing TGF-β1. Mono-cultures of MCO, MCI and MSCs served as experimental control groups. The tissue formed after 3 weeks was assessed biochemically, histochemically and by quantitative RT-PCR.
Results
Co-culture of inner (MCI) or outer (MCO) meniscus cells with MSCs resulted in neo-tissue with increased (up to 2.2-fold) proteoglycan (GAG) matrix content relative to tissues formed from mono-cultures of MSCs, MCI and MCO. Co-cultures of MCI or MCO with MSCs produced the same amount of matrix in the tissue formed. However, the expression level of aggrecan was highest in mono-cultures of MSCs but similar in the other four groups. The DNA content of the tissues from co-cultured cells was not statistically different from tissues formed from mono-cultures of MSCs, MCI and MCO. The expression of collagen I (COL1A2) mRNA increased in co-cultured cells relative to mono-cultures of MCO and MCI but not compared to MSC mono-cultures. Collagen II (COL2A1) mRNA expression increased significantly in co-cultures of both MCO and MCI with MSCs compared to their own controls (mono-cultures of MCO and MCI respectively) but only the co-cultures of MCO:MSCs were significantly increased compared to MSC control mono-cultures. Increased collagen II protein expression was visible by collagen II immuno-histochemistry. The mRNA expression level of Sox9 was similar in all pellet cultures. The expression of collagen × (COL10A1) mRNA was 2-fold higher in co-cultures of MCI:MSCs relative to co-cultures of MCO:MSCs. Additionally, other hypertrophic genes, MMP-13 and Indian Hedgehog (IHh), were highly expressed by 4-fold and 18-fold, respectively, in co-cultures of MCI:MSCs relative to co-cultures of MCO:MSCs.
Conclusions
Co-culture of primary MCI or MCO with MSCs resulted in enhanced matrix formation. MCI and MCO increased matrix formation similarly after co-culture with MSCs. However, MCO was more potent than MCI in suppressing hypertrophic differentiation of MSCs. These findings suggest that meniscus cells from the outer-vascular regions of the meniscus can be supplemented with MSCs in order to engineer functional grafts to reconstruct inner-avascular meniscus.
doi:10.1186/ar3889
PMCID: PMC3446539  PMID: 22726892
9.  Molecular Analysis of the Differentiation Potential of Murine Mesenchymal Stem Cells from Tissues of Endodermal or Mesodermal Origin 
Stem Cells and Development  2011;21(10):1761-1768.
Mesenchymal stem cells (MSCs) have received great attention due to their remarkable regenerative, angiogenic, antiapoptotic, and immunosuppressive properties. Although conventionally isolated from the bone marrow, they are known to exist in all tissues and organs, raising the question on whether they are identical cell populations or have important differences at the molecular level. To better understand the relationship between MSCs residing in different tissues, we analyzed the expression of genes related to pluripotency (SOX2 and OCT-4) and to adipogenic (C/EBP and ADIPOR1), osteogenic (OMD and ALP), and chondrogenic (COL10A1 and TRPV4) differentiation in cultures derived from murine endodermal (lung) and mesodermal (adipose) tissue maintained in different conditions. MSCs were isolated from lungs (L-MSCs) and inguinal adipose tissue (A-MSCs) and cultured in normal conditions, in overconfluence or in inductive medium for osteogenic, adipogenic, or chondrogenic differentiation. Cultures were characterized for morphology, immunophenotype, and by quantitative real-time reverse transcription–polymerase chain reaction for expression of pluripotency genes or markers of differentiation. Bone marrow–derived MSCs were also analyzed for comparison of these parameters. L-MSCs and A-MSCs exhibited the typical morphology, immunophenotype, and proliferation and differentiation pattern of MSCs. The analysis of gene expression showed a higher potential of adipose tissue–derived MSCs toward the osteogenic pathway and of lung-derived MSCs to chondrogenic differentiation, representing an important contribution for the definition of the type of cell to be used in clinical trials of cell therapy and tissue engineering.
doi:10.1089/scd.2011.0030
PMCID: PMC3376459  PMID: 21970410
10.  The effects of topical mesenchymal stem cell transplantation in canine experimental cutaneous wounds 
Veterinary Dermatology  2013;24(2):242-e53.
Background
Adult stem cells have been widely investigated in bioengineering approaches for tissue repair therapy. We evaluated the clinical value and safety of the application of cultured bone marrow-derived allogenic mesenchymal stem cells (MSCs) for treating skin wounds in a canine model.
Hypothesis
Topical allogenic MSC transplantation can accelerate the closure of experimental full-thickness cutaneous wounds and attenuate local inflammation.
Animals
Adult healthy beagle dogs (n = 10; 3–6 years old; 7.2–13.1 kg) were studied.
Methods
Full-thickness skin wounds were created on the dorsum of healthy beagles, and allogenic MSCs were injected intradermally. The rate of wound closure and the degree of collagen production were analysed histologically using haematoxylin and eosin staining and trichrome staining. The degree of cellular proliferation and angiogenesis was evaluated by immunocytochemistry using proliferating cell nuclear antigen-, vimentin- and α-smooth muscle actin-specific antibodies. Local mRNA expression levels of interleukin-2, interferon-γ, basic fibroblast growth factor and matrix metalloproteinase-2 were evaluated by RT-PCR.
Results
Compared with the vehicle-treated wounds, MSC-treated wounds showed more rapid wound closure and increased collagen synthesis, cellular proliferation and angiogenesis. Moreover, MSC-treated wounds showed decreased expression of pro-inflammatory cytokines (interleukin-2 and interferon-γ) and wound healing-related factors (basic fibroblast growth factor and matrix metalloproteinase-2).
Conclusion and clinical importance
Topical transplantation of MSCs results in paracrine effects on cellular proliferation and angiogenesis, as well as modulation of local mRNA expression of several factors related to cutaneous wound healing.
Résumé
Contexte
Les cellules souches adultes ont été largement étudiées dans les approches de bio-ingénierie pour la thérapie de réparation tissulaire. Nous évaluons l'efficacité clinique et la sécurité de l'application de cellules souches mésenchymateuses allogéniques en culture dérivées de moelle osseuse (MSCs) pour le traitement de plaies cutanées dans un modèle canin.
Hypothèse
La transplantation de MSC allogénique topique peut accélérer la fermeture en toute épaisseur de plaies cutanées expérimentales et atténuer l'inflammation locale.
Sujets
Des chiens beagles adultes sains (n = 10; 3–6 ans; 7.2–13.1 kg) ont été étudiés.
Méthodes
Des plaies cutanées en pleine épaisseur ont été crées sur la face dorsale des beagles sains et des MSCs allogènes ont été injectées par voie intradermique. Le taux de cicatrisation et le degré de production de collagène ont été analysés sur le plan histologique par colorations à l'hématoxyline et éosine et par trichrome. Le degré de prolifération cellulaire et d'angiogénèse ont été évalués par immunohistochimie à l'aide d'anticorps spécifiques d'antigène nucléaire de prolifération cellulaire, de vimentine et d'actine de muscle lisse α. Les taux d'expression local d'ARNm d'interleukine-2, d'interféron-γ, du facteur de croissance basique de fibroblaste et de métalloprotéinase-2 de matrice, ont été évalués par RT-PCR.
Résultats
Comparé avec les plaies traitées par excipient, les plaies traitées par les MSCs ont montré une plus rapide cicatrisation et une augmentation de synthèse de collagène, de prolifération cellulaire er d'angiogénèse. En outre, Les plaies traitées au MSC ont montré une expression diminuée de cytokines pro-inflammatoires (interleukine-2 et interféron-γ) et des facteurs liés à la cicatrisation (facteur de croissance basique de fibroblaste et métalloprotéinase-2 de matrice).
Conclusion et importance clinique
La transplantation topique de MSCs résulte en des effets paracrines de prolifération cellulaire et d'angiogénèse ainsi qu'en une modulation de l'expression locale d'ARNm de plusieurs facteurs liés à la cicatrisation cutanée.
Resumen
Introducción
Las células madre de adultos se han estudiado extensivamente en estrategias de bioingeniería para la reparación de tejidos. Hemos evaluado el valor clínico y la seguridad de la aplicación de células madre mesenquimales (MSCs) alogenéicas derivadas de la medula ósea para tratar las heridas de la piel en un modelo canino.
Hipótesis
El transplante tópico alogenéico de MSCs puede acelerar la cicatrización de heridas cutáneas experimentales de todo el grosor de la piel y disminuir la inflamación local.
Animales
Se estudiaron perros de raza Beagle sanos adultos (n=10; de 3-6 años de edad; 7,2-13,1 kg de peso).
Métodos
Se crearon heridas de todo el grosor de la piel en el dorso de perros Beagle sanos, y se inyectaron MSCs alogenéicas intradérmicamente. El ritmo de reparación de la herida y el grado de producción de colágeno se analizaron histológicamente utilizando tinciones de hematoxilina-eosina y tricrómico. El grado de proliferación celular y la angiogenesis se evaluaron mediante inmunohistoquímica utilizando anticuerpos específicos para el antígeno de proliferación celular nuclear (PCNA), vimentina y α-actina de músculo liso. La expresión local de mRNA para interleuquina-2 (IL-2), interferón- γ (IFN- γ), factor básico de crecimiento de fibroblastos (BFGF) y metaloproteinasa de matriz-2 se evaluaron mediante RT-PCR.
Resultados
comparado con las heridas tratadas solo con el vehículo, las heridas tratadas con MSCs mostraron una reparación más rápida y un aumento en la producción de colágeno, proliferación celular y angiogenesis. Además, las heridas tratadas con MSCs mostraron una expresión aumentada de citoquinas proinflamatorias (IL-2 e IFN- γ) y factores relacionados con la reparación de la herida (BFGF y metaloproteinasa de matriz-2).
Conclusión e importancia clínica
El transplante tópico de MSCs resulta en efectos paracrinos en la proliferación celular y angiogenesis, así como en la modulación de la expresión de mRNA de diversos factores relacionados con la cicatrización de heridas.
Zusammenfassung
Hintergrund
Adulte Stammzellen sind für ihre biotechnologische Verwendung bei der Wiederherstellung von Geweben bereits weit reichend erforscht. Wir haben die klinische Bedeutung und Sicherheit der Applikation von kultivierten allogenen mesenchymalen Stammzellen (MSCs) aus dem Knochenmark zur Behandlung von Hautwunden in einem caninen Modell evaluiert.
Hypothese
Die topische allogene MSC Transplantation kann den Wundverschluss experimenteller Hautwunden (Brandwunden Grad III) beschleunigen und die lokale Entzündung mildern.
Tiere
Erwachsene gesunde Beagles (n=10; 3-6 Jahre alt; 7,2-13,1kg) wurden in der Studie verwendet.
Methoden
Es wurden Hautwunden 3. Grades am Rücken der gesunden Beagles verursacht und allogene MSCs intradermal injiziert. Die Schnelligkeit des Wundverschlusses und das Ausmaß der Kollagenproduktion wurden histologisch mittels Hämatoxylin und Eosin Färbung und Trichromfärbung untersucht. Das Ausmaß der zellulären Proliferation und der Angiogenese wurde mittels Immunzytochemie unter Verwendung von Proliferating-Cell-Nuclear-Antigen, Vimentin und Alpha Smooth Muscle Actin-spezifischen Antikörpern evaluiert. Die lokale Exprimierung von mRNA von Interleukin-2, Interferon-γ, Basic Fibroblasten Wachstumsfaktor und Matrix Metalloproteinase-2 wurde mittels RT PCR evaluiert.
Ergebnisse
Im Vergleich zu den Wunden, die nur mit dem Trägermedium behandelt worden waren, zeigten die mit MSC-behandelten Wunden einen rascheren Verschluss und eine erhöhte Kollagensynthese, zelluläre Proliferation und Angiogenese. Darüber hinaus zeigten MSC-behandelte Wunden eine verminderte Exprimierung pro-entzündlicher Zytokine (Interleukin-2 und Interferon-γ) und Wundheilungsfaktoren (Basic Fibroblasten Wachstumsfaktor und Matrix Metalloproteinase-2).
Schlussfolgerung und klinische Bedeutung
Die topische Transplantation von MSCs resultiert in parakrinen Effekten auf die zelluläre Proliferation und Angiogenese sowie die Modulierung lokaler mRNA Exprimierung verschiedener Faktoren, die mit der Wundheilung der Haut im Zusammenhang stehen.
摘要
背景
以成体干细胞的生物工程法用于组织修复治疗已被广泛研究。将骨髓培养得到的外源性间充质干细胞(MSCs)用于犬模型上治疗皮肤创伤,我们来评估其临床价值和安全性。
假设
局部外源性MSC移植能使试验的皮肤创口全层愈合加快,并使局部炎症减轻。
动物
研究成年健康比格犬(n = 10;3–6 岁;7.2–13.1 kg)。
方法
在健康的比格犬背部创造全层皮肤创口,皮内注射外源性MSCs。使用苏木精和伊红染色法和三色染色法,从组织结构上分析伤口愈合率和胶原产生的程度。通过免疫细胞化学使用增殖细胞核抗原、维生素和α-平滑肌肌动蛋白-特定抗体,评估细胞增殖和血管生成程度。通过RT PCR评估白介素-2、γ-干扰素、基纤维母细胞生长因子和基质金属蛋白酶-2局部mRNA表达等级。
结果
与媒介物治疗创口相比,MSC治疗创口显示出更快的创口愈合,以及胶原合成、细胞增殖和血管生成增加。 此外,MSC治疗创口显示出炎性细胞因子(白介素-2和γ-干扰素)和与创口愈合相关因子(碱性成纤维细胞生长因子和基质金属蛋白酶-2)表达减少。
结论和临床价值
局部移植MSCs造成了对细胞增殖和血管生成的旁分泌作用,也同样引起几个与皮肤创伤愈合相关因子局部mRNA表达的调节。
要約
背景
成熟した幹細胞は組織修復療法に対して生物工学的なアプローチで広く研究されている。著者らは培養した骨髄由来の同種異系の間葉系肝細胞(MSCs)を犬の皮膚創傷の治療に応用し、臨床的な価値と安全性を評価した。
仮説
同種異系MSCの局所への移植は実験的な皮膚全層創傷の閉鎖と炎症の軽減を加速する可能性がある。
供与動物
成熟した健常ビーグル犬(n = 10; 3–6 歳齢; 7.2–13.1 kg)を用いた。
方法
健常ビーグルの背部に皮膚全層潰瘍を作成し、同種異系MSCを皮内に注射した。創傷閉鎖率とコラーゲン産生度をヘマトキシリン染色トリクローム染色を用いて組織学的に解析した。細胞増殖度と血管新生は核内増殖抗原−、ビメンチン−、α−平滑筋アクチン−特異的抗体を用いて免疫組織化学染色で評価した。局所のインターロイキン−2、インターフェロンγ、塩基性線維芽細胞増殖因子とマトリックスメタロプロテアーゼ2のmRNA発現レベルをRT-PCRで評価した。
結果
賦形剤のみで治療した創傷と比較し、MSCで治療した創傷はより早期に閉鎖し、コラーゲン合成の増加、細胞増殖や血管新生を示した。さらに、MSCで治療した創傷は炎症後サイトカイン(インターロイキン−2とインターフェロン−γ)と創傷治癒関連因子(塩基性線維芽細胞増殖因子とマトリックスメタロプロテアーゼ−2)の発現が減少していた。
結論と臨床的な重要性
MSCの局所への移植により細胞増殖と血管新生に対してはパラクライン効果を示すのと同時に,皮膚創傷治癒に関連する様々な因子の局所mRNA発現を調節した。
doi:10.1111/vde.12011
PMCID: PMC3618380  PMID: 23432413
11.  Umbilical cord blood-derived mesenchymal stem cells consist of a unique population of progenitors co-expressing mesenchymal stem cell and neuronal markers capable of instantaneous neuronal differentiation 
Introduction
Umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs) are self-renewing multipotent progenitors with the potential to differentiate into multiple lineages of mesoderm, in addition to generating ectodermal and endodermal lineages by crossing the germline barrier. In the present study we have investigated the ability of UCB-MSCs to generate neurons, since we were able to observe varying degrees of neuronal differentiation from a few batches of UCB-MSCs with very simple neuronal induction protocols whereas other batches required extensive exposure to combination of growth factors in a stepwise protocol. Our hypothesis was therefore that the human UCB-MSCs would contain multiple types of progenitors with varying neurogenic potential and that the ratio of the progenitors with high and low neurogenic potentials varies in different batches of UCB.
Methods
In total we collected 45 UCB samples, nine of which generated MSCs that were further expanded and characterized using immunofluorescence, fluorescence-activated cell sorting and RT-PCR analysis. The neuronal differentiation potential of the UCB-MSCs was analyzed with exposure to combination of growth factors.
Results
We could identify two different populations of progenitors within the UCB-MSCs. One population represented progenitors with innate neurogenic potential that initially express pluripotent stem cell markers such as Oct4, Nanog, Sox2, ABCG2 and neuro-ectodermal marker nestin and are capable of expanding and differentiating into neurons with exposure to simple neuronal induction conditions. The remaining population of cells, typically expressing MSC markers, requires extensive exposure to a combination of growth factors to transdifferentiate into neurons. Interesting to note was that both of these cell populations were positive for CD29 and CD105, indicating their MSC lineage, but showed prominent difference in their neurogenic potential.
Conclusion
Our results suggest that the expanded UCB-derived MSCs harbor a small unique population of cells that express pluripotent stem cell markers along with MSC markers and possess an inherent neurogenic potential. These pluripotent progenitors later generate cells expressing neural progenitor markers and are responsible for the instantaneous neuronal differentiation; the ratio of these pluripotent marker expressing cells in a batch determines the innate neurogenic potential.
doi:10.1186/scrt148
PMCID: PMC3580487  PMID: 23253356
12.  Ex vivo expanded SSEA-4+ human limbal stromal cells are multipotent and do not express other embryonic stem cell markers 
Molecular Vision  2012;18:1289-1300.
Purpose
The presence of multipotent human limbal stromal cells resembling mesenchymal stromal cells (MSC) provides new insights to the characteristic of these cells and its therapeutic potential. However, little is known about the expression of stage-specific embryonic antigen 4 (SSEA-4) and the embryonic stem cell (ESC)-like properties of these cells. We studied the expression of SSEA-4 surface protein and the various ESC and MSC markers in the ex vivo cultured limbal stromal cells. The phenotypes and multipotent differentiation potential of these cells were also evaluated.
Methods
Limbal stromal cells were derived from corneoscleral rims. The SSEA-4+ and SSEA-4- limbal stromal cells were sorted by fluorescence-activated cells sorting (FACS). Isolated cells were expanded and reanalyzed for their expression of SSEA-4. Expression of MSC and ESC markers on these cells were also analyzed by FACS. In addition, expression of limbal epithelial and corneal stromal proteins such as ATP-binding cassette sub-family G member 2 (ABCG2), tumour protein p63 (p63), paired box 6 (Pax6), cytokeratin 3 (AE5), cytokeratin 10, and keratocan sulfate were evaluated either by immunofluorecence staining or reverse transcription polymerase chain reaction. Appropriate induction medium was used to differentiate these cells into adipocytes, osteocytes, and chondrocytes.
Results
Expanded limbal stromal cells expressed the majority of mesenchymal markers. These cells were negative for ABCG2, p63, Pax6, AE-5, and keratocan sulfate. After passaged, a subpopulation of these cells showed low expression of SSEA-4 but were negative for other important ESC surface markers such as Tra-1–60, Tra-1–81, and transcription factors like octamer-binding transcription factor 4 (Oct4), SRY(sex determining region Y)-box 2 (Sox2), and Nanog. Early passaged cells when induced were able to differentiate into adipocytes, osteocytes and chondrocytes.
Conclusions
The expanded limbal stromal cells showed features of multipotent MSC. Our study confirmed the expression of SSEA-4 by a subpopulation of cultured limbal stromal cells. However, despite the expression of SSEA-4, these cells did not express any other markers of ESC. Therefore, we conclude that the cells did not show properties of ESC.
PMCID: PMC3365132  PMID: 22665977
13.  Cardiogenic and Myogenic Gene Expression in Mesenchymal Stem Cells After 5-Azacytidine Treatment 
Turkish Journal of Hematology  2013;30(2):115-121.
Objective: 5-Azacytidine is a hypomethylating agent that is used for the treatment of myelodysplastic syndrome. This histone modifier is widely employed and plays a nonspecific role in influencing the differentiation capability of stem cells. The ability of bone marrow mesenchymal stem cells to differentiate into cardiomyocyte- and myocyte-like cells after exposure to 3 different doses of 5-azacytidine has been evaluated and compared. The aim of the study was to optimize the effective dose of 5-azacytidine for promoting the cardiomyocyte and myocyte differentiation capabilities of human mesenchymal stem cells (MSCs).
Materials and Methods: Human bone marrow aspirations were collected from healthy donors. MSCs were used for the study of mesodermal differentiation. MSCs were cultured to promote osteoblast differentiation and adipocyte differentiation. The evaluation of osteogenic or adipogenic properties was then performed through immunocytochemical staining. BMMSCs were trypsinized into single-cell suspensions and then prepared for flow cytometric analysis. The MSCs were treated with 5, 10, or 15 μM 5-azacytidine for 24 h and then cultured for 3 weeks. Total RNA was extracted from untreated and 5-azacytidine–treated cells. Troponin T and GATA4 antibodies were used as cardiogenic markers, whereas myogenin and MyoD antibodies were used as myocyte markers.
Results: The morphology and growth rate of MSCs that were treated with any of the 3 doses of 5-azacytidine were similar to the morphology and growth rate of control MSCs. An immunofluorescence analysis examining the expression of the cardiac-specific markers GATA4 and troponin T and the skeletal muscle-specific markers MyoD and myogenin revealed that cells treated with 15 μM 5-azacytidine were strongly positive for these markers. Real-time RT-PCR results were examined; these amplifications indicated that there were higher expression levels of cardiac- and skeletal muscle-specific mRNAs in MSCs treated with 15 μm 5-azacytidine than in MSCs that had either been treated with lower doses of 5-azacytidine or left untreated.
Conclusion: MSCs treated with 5-azacytidine demonstrated the capacity to differentiate into both cardiomyocytes and skeletal myocytes, and 15 μM 5-azacytidine could be the optimal dose of this drug. Other promoting factors should be examined to investigate the possibility of promoting the differentiation of MSCs into specific cell types.
Conflict of interest:None declared.
doi:10.4274/Tjh.2012.0161
PMCID: PMC3878477  PMID: 24385773
mesenchymal stem cells; differentiation; Cardiomyocyte; Myocyte
14.  Characteristics and neural-like differentiation of mesenchymal stem cells derived from foetal porcine bone marrow 
Bioscience Reports  2013;33(2):e00032.
MSCs (mesenchymal stem cells) are a stem cell source that can be easily obtained from bone marrow. Despite the increasing importance of the pig as a large animal model, little is known about foetal pMSCs (porcine MSCs). In this study, we observed the gene expression of pluripotent markers in foetal pMSCs and the capacity of pMSCs to differentiate into adipocytes, osteocytes and neural-like cells using quantitative RT–PCR (reverse transcription–PCR), normal histological staining and immunohistochemistry. Foetal pMSCs have either a spindle or a flattened shape, and flow cytometry revealed the expression of the MSC-related proteins CD44 and CD105 (endoglin) but not CD34 and CD45. pMSCs express pluripotent markers such as Oct4 (octamer-binding transcription factor 4) and Nanog at the protein and mRNA levels. qRT-PCR (quantitative real-time PCR) analyses revealed that pMSCs expressed nestin [for NSCs (neural stem cells)]. Immunocytochemical and RT–PCR data showed that 29% and 23% of pMSCs expressed MAP2 (microtubule-associated protein 2) for neurons and β-tubulin III (Tuj1) for immature neurons, respectively, after induction of neural differentiation. These findings demonstrate the plasticity of pMSCs and their potential for use in cellular replacement therapy for neural diseases.
doi:10.1042/BSR20120023
PMCID: PMC3610297  PMID: 23458182
differentiation; mesenchymal stem cell; octamer-binding transcription factor 4; porcine bone marrow; reverse transcription–PCR; bFGF, basic fibroblast growth factor; COC, cumulus–oocyte complex; DMEM, Dulbecco’s modified Eagle’s medium; EGF, epidermal growth factor; ESC, embryonic stem cell; FBS, foetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IVM, in vitro maturation; MAP2, microtubule-associated protein 2; MSC, mesenchymal stem cell; NSC, neural stem cell; Oct4, octamer-binding transcription factor 4; PB, parthenogenetic blastocyst; PI, propidium iodide; pMSC, porcine MSC; PPARG, peroxisome-proliferator-activated receptor γ; PVA, poly(vinyl alcohol); qRT-PCR, quantitative real-time PCR; RA, retinoic acid; RT–PCR, reverse transcription–PCR; TGFβ, transforming growth factor β
15.  Human Embryonic and Fetal Mesenchymal Stem Cells Differentiate toward Three Different Cardiac Lineages in Contrast to Their Adult Counterparts 
PLoS ONE  2011;6(9):e24164.
Mesenchymal stem cells (MSCs) show unexplained differences in differentiation potential. In this study, differentiation of human (h) MSCs derived from embryonic, fetal and adult sources toward cardiomyocytes, endothelial and smooth muscle cells was investigated. Labeled hMSCs derived from embryonic stem cells (hESC-MSCs), fetal umbilical cord, bone marrow, amniotic membrane and adult bone marrow and adipose tissue were co-cultured with neonatal rat cardiomyocytes (nrCMCs) or cardiac fibroblasts (nrCFBs) for 10 days, and also cultured under angiogenic conditions. Cardiomyogenesis was assessed by human-specific immunocytological analysis, whole-cell current-clamp recordings, human-specific qRT-PCR and optical mapping. After co-culture with nrCMCs, significantly more hESC-MSCs than fetal hMSCs stained positive for α-actinin, whereas adult hMSCs stained negative. Furthermore, functional cardiomyogenic differentiation, based on action potential recordings, was shown to occur, but not in adult hMSCs. Of all sources, hESC-MSCs expressed most cardiac-specific genes. hESC-MSCs and fetal hMSCs contained significantly higher basal levels of connexin43 than adult hMSCs and co-culture with nrCMCs increased expression. After co-culture with nrCFBs, hESC-MSCs and fetal hMSCs did not express α-actinin and connexin43 expression was decreased. Conduction velocity (CV) in co-cultures of nrCMCs and hESC-MSCs was significantly higher than in co-cultures with fetal or adult hMSCs. In angiogenesis bioassays, only hESC-MSCs and fetal hMSCs were able to form capillary-like structures, which stained for smooth muscle and endothelial cell markers.Human embryonic and fetal MSCs differentiate toward three different cardiac lineages, in contrast to adult MSCs. Cardiomyogenesis is determined by stimuli from the cellular microenvironment, where connexin43 may play an important role.
doi:10.1371/journal.pone.0024164
PMCID: PMC3170333  PMID: 21931658
16.  Comparison of phenotypic markers and neural differentiation potential of multipotent adult progenitor cells and mesenchymal stem cells 
World Journal of Stem Cells  2013;5(2):53-60.
AIM: To compare the phenotypic and neural differentiation potential of human bone marrow derived multipotent adult progenitor cells (MAPC) and mesenchymal stem cells (MSC).
METHODS: Cultures of MAPC and MSC were established in parallel from same samples of human bone marrow (n = 5). Both stem cell types were evaluated for expression of pluripotency markers including Oct-4 and Nanog by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) and expression of standard mesenchymal markers including CD14, CD34, CD44, CD45, CD73, CD90, CD105 and human leukocyte antigen (HLA)-ABC by flow cytometry. After treatment with neural induction medium both MAPC and MSC were evaluated for expression of neural proteins [neuronal filament-200 (NF-200) and glial fibrillar acidic protein (GFAP)] by immunocytochemistry and Western blotting and neural genes [NF-200, GFAP, Tau, microtubule-associated protein (MAP)-1B, MAP-2, neuron-specific enolase (NSE) and oligodendrocyte-1 (Olig-1)] by quantitative real-time-PCR.
RESULTS: MAPC had small trigonal shaped while MSC had elongated spindle-shaped morphology. The MAPC expressed Oct-4 and Nanog both at gene and protein levels, whereas MSC were negative for these pluripotent markers. MAPC were negative for HLA-ABC while MSC had high expression of HLA-ABC. In addition, MAPC as compared to MSC had significantly lower expression of CD44 (36.56% ± 1.92% vs 98.23% ± 0.51%), CD73 (15.11% ± 2.24% vs 98.53% ± 2.22%) and CD105 (13.81% ± 3.82% vs 95.12% ± 5.65%) (P < 0.001, for all) MAPC cultures compared to MSC cultures treated with neural induction medium had significantly higher fold change expression of NF-200 (0.64), GFAP (0.52), Tau (0.59), MAP-2 (0.72), Olig-1 (0.18) and NSE (0.29) proteins (P < 0.01 for Olig-1 and P < 0.001 for rest) as well as higher fold change expression of genes of NF-200 (1.34), GFAP (1.12), Tau (1.08), MAP-1B (0.92), MAP-2 (1.14) and NSE (0.4) (P < 0.001 for all).
CONCLUSION: MAPC can be differentially characterized from MSC as Oct-4 and Nanog positive stem cells with no expression of HLA-ABC and low expression of mesenchymal markers CD44, CD73 and CD105 and when compared to MSC they possess greater predilection for differentiation into neuro-ectodermal lineage.
doi:10.4252/wjsc.v5.i2.53
PMCID: PMC3648646  PMID: 23671719
Bone marrow; Human multipotent adult progenitor cells; Human mesenchymal; Stem cells; Phenotypic markers; Neural differentiation
17.  Mesenchymal cells from limbal stroma of human eye 
Molecular Vision  2008;14:431-442.
Purpose
Mesenchymal stem cells (MSC) are self-renewing, multipotent cells that are present in many adult tissues, including bone marrow, trabecular bone, adipose, and muscle. The presence of such cells of mesenchymal origin and their role during the wound healing of ocular injuries are currently being explored by many studies worldwide. In this study, we aimed to report the presence of mesenchymal cells resembling bone marrow-derived cells (MSC-BM) in the limbus of the human eye.
Methods
Fresh limbal tissues obtained from human subjects undergoing limbal biopsy for ocular surface reconstruction were used to establish limbal mesenchymal cell (MC-L) cultures. The spindle cell outgrowths observed in extended limbal epithelial cultures (LECs) and from deepithelialized limbal tissues were serially passaged using a human corneal epithelial (HCE) medium, which contained epidermal growth factor (EGF) and insulin, and supplemented with fetal bovine serum (FBS). MSC cultures were established from human bone marrow samples using Dulbecco’s Modified Eagles Medium (DMEM) supplemented with FBS. The mesenchymal cells from both extended limbal cultures (MC-L) and bone marrow (MSC-BM) were characterized by morphology and immunophenotyping using epithelial, mesenchymal, hematopoietic, and endothelial markers using fluorescent-activated cell sorting (FACS). Selective markers were further confirmed by immunostaining and reverse transcription polymerase chain reaction (RT–PCR). Stromal cells of both origins (limbal and bone marrow-derived) were also evaluated for colony forming ability and population doubling. Attempts were made to differentiate these into adipocytes and osteocytes using conditioned medium.
Results
Spindle cells from extended limbal epithelial cultures as well as de-epithelialized human limbal tissues appeared elongated and fibroblast-like with oval vesicular nuclei. Both MC-L and MSC-BM showed colony forming ability in 14 days of plating. MC-L showed a population doubling of 22.95 while in MSC-BM, it was 30.98. Immunophenotyping of these cells by FACS and immunocytochemistry showed that the MC-L were positive for mesenchymal markers and negative for epithelial and hematopoietic markers similar to MSC-BM. The MC-L phenotype has thus been defined as MC-LCD105, CD106, CD54, CD166, CD90, CD29, CD71, pax −6 +/ p75, SSEA1, Tra-1–61, Tra-1–81, CD31, CD34, CD45, CD11a, CD11c, CD14, CD138, Flk1, Flt1, VE-Cadherin -. The profile was further confirmed by RT–PCR. These cells also showed differentiation into adipocytes and osteocytes.
Conclusions
We demonstrated the presence of mesenchymal cells in the human limbus, similar to the bone marrow-derived MSC-BM. This presence suggests that these cells are unique to the adult stem cell niche.
PMCID: PMC2268845  PMID: 18334960
18.  Development and Characterization of a Clinically Compliant Xeno-Free Culture Medium in Good Manufacturing Practice for Human Multipotent Mesenchymal Stem Cells 
Stem Cells Translational Medicine  2012;1(10):750-758.
This study developed and tested a serum-free and xeno-free culture medium (SFM-XF) using human bone marrow- and adipose-derived mesenchymal stem cells (MSCs). Human MSCs expanded in SFM-XF showed continual propagation, with an expected phenotype and differentiation potential to adipogenic, chondrogenic, and osteogenic lineages similar to that of traditional serum-containing culture medium (SCM). The SFM-XF culture system allows better expansion and multipotentiality of MSCs and serves as a preferred alternative to SCM for the production of large scale, functionally competent MSCs for future clinical applications.
Human multipotent mesenchymal stem cell (MSC) therapies are currently being tested in clinical trials for Crohn's disease, multiple sclerosis, graft-versus-host disease, type 1 diabetes, bone fractures, cartilage damage, and cardiac diseases. Despite remarkable progress in clinical trials, most applications still use traditional culture media containing fetal bovine serum or serum-free media that contain serum albumin, insulin, and transferrin. The ill-defined and variable nature of traditional culture media remains a challenge and has created a need for better defined xeno-free culture media to meet the regulatory and long-term safety requirements for cell-based therapies. We developed and tested a serum-free and xeno-free culture medium (SFM-XF) using human bone marrow- and adipose-derived MSCs by investigating primary cell isolation, multiple passage expansion, mesoderm differentiation, cellular phenotype, and gene expression analysis, which are critical for complying with translation to cell therapy. Human MSCs expanded in SFM-XF showed continual propagation, with an expected phenotype and differentiation potential to adipogenic, chondrogenic, and osteogenic lineages similar to that of MSCs expanded in traditional serum-containing culture medium (SCM). To monitor global gene expression, the transcriptomes of bone marrow-derived MSCs expanded in SFM-XF and SCM were compared, revealing relatively similar expression profiles. In addition, the SFM-XF supported the isolation and propagation of human MSCs from primary human marrow aspirates, ensuring that these methods and reagents are compatible for translation to therapy. The SFM-XF culture system allows better expansion and multipotentiality of MSCs and serves as a preferred alternative to serum-containing media for the production of large scale, functionally competent MSCs for future clinical applications.
doi:10.5966/sctm.2012-0072
PMCID: PMC3659659  PMID: 23197667
Bone marrow stromal cells; Cellular therapy; Chondrogenesis; Culture; Differentiation; Marrow stromal stem cells; Microarray; Serum-free medium
19.  Effect of Recombinant Human Erythropoietin On the Stemness of Bone Marrow-derived Mesenchymal Stem Cells in vitro 
The purpose of this study was to investigate the effects of the recombinant human erythropoietin (rhEPO) on proliferative and multi-differentiation potential of the bone marrow-derived mesenchymal stem cells (MSCs). The MSCs were isolated primarily from bone marrow of adult rat and purified at increasing passage. A purified population of MSCs can be obtained about 2 weeks after the initiation of culture. After three passages (P3-MSCs), bone marrow-derived adherent cells were identified, then different concentrations of rhEPO (0.1, 1, 5, 10, 100 U/ml) was added into the Passage-3 cells which had been identified. The expression of the surface markers in adherent cells was detected by the flow cytometry. The mRNA levels of transcription factors OCT4, SOX2, Nanog and TERT were measured by reverse transcription-polymerase chain reaction (RT-PCR). The results showed that CD29 and CD90 were positive in MSCs, but not CD33, CD44 and CD45, and the cells could differentiate into multiple lineages such as osteocytes and adipocytes. The expression of OCT4, SOX2, TERT, Nanog mRNA were up-regulated by the treatment of EPO. The effect of EPO was the most obvious when its concentration was 5U/mL after 12h. we conclude that MSCs can not only perserve characteristics of stem cells but also maintain its multi-lineage differentiation potential after appropriate treatment of EPO.
PMCID: PMC4021811  PMID: 24855555
Erythropoietin; Mesenchymal stem cells; Differentiation potential
20.  Comparison of proliferative and multilineage differentiation potentials of cord matrix, cord blood, and bone marrow mesenchymal stem cells 
Background:
Hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) are the two widely studied and characterized adult stem cells. Thus far, MSCs were obtained from the bone marrow, which is a painful procedure. Therefore, MSCs from less common sources like cord blood, adipose tissue, tooth pulp, and so on, have been the subject of research. The purpose of this study is to explore the possibility of finding MSCs from a less controversial, easy, and abundant source, such as the umbilical cord, for potential regenerative medicine applications.
Study Design and Methods:
Five bone marrow samples (BM), seventy cord blood units (CB), and four umbilical cord matrix (CM) samples have been used for the study. Expanded MSCs were checked for biomarker expression by flow cytometry and were also checked for their differentiation to mesodermal and ectodermal lineages.
Results:
MSCs could be isolated from 100% BM and CM samples, as compared to only 6% of CB samples. The fold expansion of the mesenchymal stem cells observed in CB (CB-MSCs) was distinctly higher as compared to BM (BM-MSCs) and CM (CM-MSCs). MSCs isolated from all the three sources expressed a characteristic mesenchymal phenotype of CD45 − /vWF − /CD14 − /CD31 − /CD73 + /CD105 + /SSEA4 + /CD29 + /CD44 + /HLAABC +, whereas, the HLA DR was conspicuously absent in CM-MSCs and CB-MSCs. Although osteogenic, chondrogenic, and neural differentiation was observed in MSCs from all sources, adipogenic differentiation was observed only in BM-MSCs.
Conclusion:
CM-MSCs are a dependable source of an unlimited number of MSCs for autologous and allogenic use in regenerative medicine.
doi:10.4103/0973-6247.59386
PMCID: PMC2847339  PMID: 20376261
Mesenchymal stem cells; bone marrow; umbilical cord blood; umbilical cord
21.  Comprehensive Characterization of Mesenchymal Stem Cells from Human Placenta and Fetal Membrane and Their Response to Osteoactivin Stimulation 
Stem Cells International  2012;2012:658356.
Mesenchymal stem cells (MSCs) are the most promising seed cells for cell therapy and can be isolated from various sources of human adult tissues such as bone marrow (BM-MSC) and adipose tissue. However, cells from these tissues must be obtained through invasive procedures. We, therefore, characterized MSCs isolated from fresh placenta (Pl-MSC) and fetal membrane (Mb-MSC) through morphological and fluorescent-activated cell sorting (FACS). MSC frequency is higher in membrane than placenta (2.14%  ± 0.65 versus 15.67%  ± 0.29%). Pl/Mb-MSCs in vitro expansion potential was significantly higher than BM-MSCs. We demonstrated that one of the MSC-specific marker is sufficient for MSC isolation and that culture in specific media is the optimal way for selecting very homogenous MSC population. These MSCs could be differentiated into mesodermal cells expressing cell markers and cytologic staining consistent with mature osteoblasts and adipocytes. Transcriptomic analysis and cytokine arrays demonstrated broad similarity between placenta- and membrane-derived MSCs and only discrete differences with BM-MSCs with enrichment of networks involved in bone differentiation. Pl/Mb-MSCs displayed higher osteogenic differentiation potential than BM-MSC when their response to osteoactivin was evaluated. Fetal-tissue-derived mesenchymal cells may, therefore, be considered as a major source of MSCs to reach clinical scale banking in particular for bone regeneration.
doi:10.1155/2012/658356
PMCID: PMC3373208  PMID: 22701494
22.  In vitro Effects of RU486 on Proliferation and Differentiation Capabilities of Human Bone Marrow Mesenchymal Stromal Cells 
Steroids  2011;77(1-2):132-137.
Although exogenous glucocorticoids (GC) play a role in the regulation of bone marrow mesenchymal stem/stromal cells (MSCs) proliferation and differentiation, the function of endogenous GC is not well understood. The purpose of this study was to investigate the effect of the blockage of endogenous GC using RU486, an antagonist of the glucocorticoid receptor, on the in vitro proliferation and differentiation capabilities of human MSCs. We quantitatively measured cell proliferation of human MSCs after treatment with increasing concentrations of RU486. We also evaluated multiple MSC differentiation capabilities, as well as the expression of stemness and senescence genes after proliferation of these human cells in vitro in the presence of RU486 at 10−8M. It was observed that RU486 treatment significantly increases the proliferation of human MSCs, although the optimal dose of RU486 for this increase in proliferation differs depending on the gender of the MSC donor. This improvement in MSC proliferation with RU486 treatment was higher in MSCs from male donors than that from females. No effect of RU486 on MSC proliferation was observed in a steroid-free medium. RU486 pretreatment significantly increased the expression of mRNA for alkaline phosphatase in human MSCs and the mRNA expression of osteocalcin of these cells up-regulated earlier after their exposure to osteogenic differentiation medium. Although no statistical significance in terms of chondrogenic differentiation markers was detected, mRNA expression for aggrecan and collagen type 2 were higher in a majority of the RU486-pretreated donor MSCs than their untreated controls. No significant difference in terms of MSC adipogenic differentiation capabilities were observed after RU486 treatment. RU486 treatment up-regulated the expressions of FGF-2 and Sox-11 in human MSCs. These results indicate that blockage of endogenous GCs may be developed as a novel approach to effectively improve the proliferation and osteochondral differentiation capabilities of human MSCs for potential clinical applications. Additional studies will be required to determine the potential long-term effects of RU486 treatment on these bone marrow cells.
doi:10.1016/j.steroids.2011.10.017
PMCID: PMC3242919  PMID: 22093480
Glucocorticoids; Antagonist; Bone marrow mesenchymal stem/stromal cells; Proliferation; Differentiation; Senescence
23.  Induced Pluripotent Stem Cells from Human Hair Follicle Mesenchymal Stem Cells 
Stem Cell Reviews  2012;9(4):451-460.
Reprogramming of somatic cells into inducible pluripotent stem cells (iPSCs) provides an alternative to using embryonic stem cells (ESCs). Mesenchymal stem cells derived from human hair follicles (hHF-MSCs) are easily accessible, reproducible by direct plucking of human hairs. Whether these hHF-MSCs can be reprogrammed has not been previously reported. Here we report the generation of iPSCs from hHF-MSCs obtained by plucking several hairs. hHF-MSCs were isolated from hair follicle tissues and their mesenchymal nature confirmed by detecting cell surface antigens and multilineage differentiation potential towards adipocytes and osteoblasts. They were then reprogrammed into iPSCs by lentiviral transduction with Oct4, Sox2, c-Myc and Klf4. hHF-MSC-derived iPSCs appeared indistinguishable from human embryonic stem cells (hESCs) in colony morphology, expression of alkaline phosphotase, and expression of specific hESCs surface markers, SSEA-3, SSEA-4, Tra-1-60, Tra-1-81, Nanog, Oct4, E-Cadherin and endogenous pluripotent genes. When injected into immunocompromised mice, hHF-MSC-derived iPSCs formed teratomas containing representatives of all three germ layers. This is the first study to report reprogramming of hHF-MSCs into iPSCs.
Electronic supplementary material
The online version of this article (doi:10.1007/s12015-012-9420-5) contains supplementary material, which is available to authorized users.
doi:10.1007/s12015-012-9420-5
PMCID: PMC3742959  PMID: 23242965
Induced pluripotent stem cells; Mesenchymal stem cells; Human hair follicle; Reprogrammed
24.  Isolation and Characterization of Porcine Amniotic Fluid-Derived Multipotent Stem Cells 
PLoS ONE  2011;6(5):e19964.
The aim of this study was to isolate and characterize porcine amniotic fluid-derived multipotent stem cells (pAF-MSC). The porcine amniotic fluid (AF) from the amniotic cavity of pregnant gilts in the early stages of gestation (at E35) was collected and centrifuged for 5–10 min at 400 g to pellet cells. The primary culture of AF showed the multiple cell types, including the epithelial-like cells and fibroblast-like cells. By culturing in AMM medium for 6 to 8 days, the epithelial-like cells disappeared and the remaining cells presented the fibroblastoid morphology. The doubling time of pAF-MSCs was about 34.6 h, and the cells had been continually cultured over 60 passages in vitro. The flow cytometry results showed that pAF-MSCs were positive for CD44, CD117 and CD166, but negative for CD34, CD45 and CD54. Meanwhile, pAF-MSCs expressed ES cell markers, such as Oct4, Nanog, SSEA4, Tra-1-60 and Tra-1-81. The ratio of CD117+ CD44+ cells accounted for 98% of pAF-MSCs population. Three germ layer markers, including FGF5 (ectodermal marker), AFP (endodermal marker) and Bra (mesodermal marker), were detected in embryoid bodies derived from pAF-MSCs. Under the different induction conditions, the pAF-MSCs were capable of differentiating into neurocytes, adipocytes and beating cardiomyocytes. Furthermore, the pAF-MSCs didn't form teratoma when injected into immunodeficiency mice. These optimal features of pAF-MSCs provide an excellent alternative stem cell resource for potential cell therapy in regenerative medicine and transgenic animals.
doi:10.1371/journal.pone.0019964
PMCID: PMC3098286  PMID: 21625520
25.  Human Dermis Harbors Distinct Mesenchymal Stromal Cell Subsets 
Multipotent mesenchymal stromal cells (MSCs) are found in a variety of adult tissues including human dermis. These MSCs are morphologically similar to bone marrow–derived MSCs, but are of unclear phenotype. To shed light on the characteristics of human dermal MSCs, this study was designed to identify and isolate dermal MSCs by a specific marker expression profile, and subsequently rate their mesenchymal differentiation potential. Immunohistochemical staining showed that MSC markers CD73/CD90/CD105, as well as CD271 and SSEA-4, are expressed on dermal cells in situ. Flow cytometric analysis revealed a phenotype similar to bone marrow–derived MSCs. Human dermal cells isolated by plastic adherence had a lower differentiation capacity as compared with bone marrow–derived MSCs. To distinguish dermal MSCs from differentiated fibroblasts, we immunoselected CD271+ and SSEA-4+ cells from adherent dermal cells and investigated their mesenchymal differentiation capacity. This revealed that cells with increased adipogenic, osteogenic, and chondrogenic potential were enriched in the dermal CD271+ population. The differentiation potential of dermal SSEA-4+ cells, in contrast, appeared to be limited to adipogenesis. These results indicate that specific cell populations with variable mesenchymal differentiation potential can be isolated from human dermis. Moreover, we identified three different subsets of dermal mesenchymal progenitor cells.
doi:10.1038/jid.2011.355
PMCID: PMC3278768  PMID: 22048731

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