Gene Expression In Mesenchymal Stem Cells Research Articles

The Effect of Link N on the Differentiation of Human Mesenchymal Stem Cells

Introduction The degeneration of intervertebral disk (IVD) can cause low back pain, and invoking the regeneration of degenerated IVD can relieve the pain.1,2 Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into chondrocytes and can be employed in cartilage regeneration.3,4 Link N (DHLSDNYTLDHDRAIH) is the amino terminal peptide of link protein in the extracellular matrix of cartilage. It can stimulate the biosynthesis of collagens II and IX and aggrecan in IVD cells in vitro as well as in vivo.5–7 We hypothesized that Link N can enhance the chondrogenic differentiation of MSCs, and can be applied with MSCs in bio-regeneration of IVDs. The specific aim of this research is to determine the effect of Link N on gene expression of MSCs. Materials and Methods MSCs were isolated from the bone marrow of osteoarthritis (OA) patients. The cells were cultured in 24-well plates (3000 cells/well) in chondrogenesis differentiation medium (Invitrogen, Canada) according to the manufacturer's instructions. Link N was dissolved in the media with a final concentration of 0.1 µg/mL and 1 µg/mL, respectively. Medium without Link N was applied as a control. The media were changed every 3 days, and the used media were collected and stored at −20°C for GAG analysis. For gene expression analysis, the cells were cultured for 7, 14, and 21 days, then were washed twice with PBS and dissolved in Trizol (Invitrogen, Canada). Total RNA was isolated and cDNA was synthesized by reverse transcription. Gene expression was analyzed by real-time PCR and relative expression was calculated using GAPDH as a reference gene. MSCs from three OA patients were analyzed and statistical significance was calculated using analysis of variance followed by Fisher protected least significant difference (PLSD) comparison test. Results With the concentrations of 0.1 and 1.0 µg/mL, no toxic effect of Link N on MSCs was observed. After MSCs were cultured for 7 days and 14 days, the expression of aggrecan (ACAN) (Fig. 1), collagen II (COL2A1) (Fig. 2) and the transcription regulator SOX9 increased significantly in the media with 0.1 µg/mL or 1.0 µg/mL Link N. No significant differences were observed at day 21 (Figs. 1 and 2). When cells were cultured with 0.1 or 1.0 µg/mL Link N, the quantity of GAG in the media increased significantly at day 9, 12, and 15, but no major difference was observed at day 3 and 6 and at day 18 and 21. When MSCs were cultured for 7, 14, and 21 days, no significant effect of Link N on the expression of alkaline phosphatase (ALP) was observed. Furthermore, Link N significantly inhibited osteocalcin (OC) gene expression after culturing MSCs for 21 days. Conclusion Link N can improve the chondrogenic differentiation of MSCs and inhibit their osteogenic differentiation. It can therefore be used in conjunction with MSCs in the repair of IVD degeneration. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Luoma K, Riihimaki H, Luukkonen R, Raininko R, Viikari-Juntura E, Lamminen A. Low back pain in relation to lumbar disk degeneration. Spine 2000;25:487–492 Roughley PJ. Biology of intervertebral disk aging and degeneration: involvement of the extracellular matrix. Spine 2004;29:2691–2699 Xian CJ, Foster BK. Repair of injured articular and growth plate cartilage using mesenchymal stem cells and chondrogenic gene therapy. Current Stem Cell Research Therapy 2006;1:213–229 Koga H, Engebretsen L, Brinchmann JE, Muneta T, Sekiya I. Mesenchymal stem cell-based therapy for cartilage repair: a review. Knee Surgery Sports Traumatol Arthroscopy 2009;7:1289–1297 Mwale F, Demers CN, Petit A et al. A synthetic peptide of link protein stimulates the biosynthesis of collagens II, IX and proteoglycan by cells of the intervertebral disc. Journal of Cell Biochemistry 2003;88:1202–1213 Petit A, Yao G, Rowas SA, et al. Effect of synthetic Link N peptide on the type I and type II collagens in human intervertebral discs cells. Tissue Engeneering Part A 2011;17:899–904

Thermoreversible Hyaluronan Hydrogels Support Mesenchymal Stem Cells Discogenic Differentiation in Vitro and Ex Vivo

Introduction Hyaluronan is a proteoglycan widely present in the nucleus pulposus (NP) of the intervertebral disk (IVD). Recently, thermoreversible hyaluronan (HA) based hydrogels, which are injectable, minimally invasive, allow for encapsulation and maintenance of viable cells and promote re-differentiation of NP cells, have been developed.1 While it is known that hyaluronan is recognized by mesenchymal stem cells (MSCs)2 and can induce anabolic pathways, growth factors may further support MSC differentiation in HA-based hydrogels. Therefore, the objectives of the present work were: (i) to assess whether the addition of growth factors3 could support MSC differentiation towards the disk cell phenotype in HA based hydrogels and (ii) to determine if MSC preconditioning (culture under “chondrogenic” conditions) would improve the survival rate and differentiation potential of MSC supplemented to an IVD cultured ex vivo. Materials and Methods Hydrogel Thermoreversible HA-based hydrogels were prepared by grafting a thermoreversible segment poly (N-isopropylacrylamide) (pNIPAM) on a functionalized HA backbone, as previously described.1 In vitro Human bone marrow-derived MSCs (P2-P4) were cultured for 7 days in HA-pNIPAM hydrogel under hypoxic conditions (5% O2) in chondrogenic medium, with or without recombinant human growth and differentiation factor-5 (rhGDF-5) supplementation. Cell viability, DNA, and glycosaminoglycans (GAG) content were quantified by Live-dead, Picogreen, and 1,9 dimethylmethylene blue assays, respectively. Gene expression analysis was performed to assess the disc-like differentiation of MSCs. The expression of collagen type I and II (COL1 and COL2) aggrecan (ACAN), cytokeratin-19, sox-9, cluster of differentiation 24 (CD24), hyaluronan synthase I and II (HAS1 and HAS2), forkhead box protein F1 (FOXF1) and carbonic anhydrase 12 (CA12) was analyzed with 18s as endogenous control. Ex Vivo Bovine caudal IVDs were excised and nucleotomy performed through the endplate (Fig. 1A,B). MSCs were either suspended in HA-pNIPAM and directly supplied to the IVDs or precultured (under hypoxic conditions and with supplementation of GDF-5) for 1 week in HA-pNIPAM and then supplied to the IVDs. After 1 week of culture, gene expression analyses were performed using the same markers as for the in vitro study. Statistics After confirming the normality of the data distribution, one-way analysis of variance with Bonferroni posthoc was performed ( p < 0.05). Results MSC viability was maintained>90% over 1 week of culture in the HA-pNIPAM hydrogel both in vitro and ex vivo. DNA and GAG did not show significant variation over the 7 days of in vitro culture. Gene expression revealed 10–100-fold upregulation of the transcription factor SOX9 and 100–1000, fold upregulation of COL2, while COL1 was only slightly enhanced. The expression of the potential NP cell marker KRT19 was also increased. With the addition of rhGDF-5, an improved COL2/COL1 expression ratio was found. No significant differences were found for the other genes. Ex vivo cultures of nucleotomized discs supplemented with MSCs in HA-pNIPAM confirmed the positive effect of the hydrogel and the IVD environment on MSC differentiation towards the disc-like phenotype (Fig. 1C), with upregulation of collagen type II ( p < 0.05), CD24, and FOX-F1. No significant differences were found for the other genes. Preculture of MSCs before the supplementation in the IVD did not enhance MSC differentiation. Figure 1 Bovine IVD after nucleotomy: (A) top view and (B) view of the nucleotomized area. (C) Gene expression of human MSCs after 1 week of culture in bovine IVDs normalized to the mRNA levels at day 0 of culture: human MSCs were either suspended in HA-pNIPAM and directly supplied to bovine IVDs (white bars) or precultured for 1 week in HA-pNIPAM hydrogel and then supplied to bovine IVDs (grey bars);* p < 0.05, outliers. Conclusion The thermoreversible hyaluronan-based hydrogel was able to induce human MSC differentiation toward a “disc-like” phenotype without the addition of growth factors, although GDF-5 supplementation resulted in an improved gene expression profile. Preculture of MSCs before the supplementation in the IVD is not required with the thermoreversible hyaluronan-based hydrogel. Therefore, this type of hydrogel could be an appropriate carrier for MSCs supplementation to compromised discs. Investigations are ongoing to evaluate its biomechanical behavior under loading. Acknowledgements This study was partially supported by a NASS Research Grant. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Peroglio M, et al. European Spine Journal (Epub ahead of print) Zhu H et al. Stem cells 2006; 24:928–935 Gantenbein-Ritter B, et al. European Spine Journal 2011;20(6):962–971

Unsaturated fatty acids induce mesenchymal stem cells to increase secretion of angiogenic mediators

Mesenchymal stem cells (MSC) represent emerging cell-based therapies for diabetes and associated complications. Ongoing clinical trials are using exogenous MSC to treat type 1 and 2 diabetes, cardiovascular disease and non-healing wounds due to diabetes. The majority of these trials are aimed at exploiting the ability of these multipotent mesenchymal stromal cells to release soluble mediators that reduce inflammation and promote both angiogenesis and cell survival at sites of tissue damage. Growing evidence suggests that MSC secretion of soluble factors is dependent on tissue microenvironment. Despite the contribution of fatty acids to the metabolic environment of type 2 diabetes, almost nothing is known about their effects on MSC secretion of growth factors and cytokines. In this study, human bone marrow-derived MSC were exposed to linoleic acid, an omega-6 polyunsaturated fatty acid, or oleic acid, a monounsaturated fatty acid, for seven days in the presence of 5.38 mM glucose. Outcomes measured included MSC proliferation, gene expression, protein secretion and chemotaxis. Linoleic and oleic acids inhibited MSC proliferation and altered MSC expression and secretion of known mediators of angiogenesis. Both unsaturated fatty acids induced MSC to increase secretion of interleukin-6, VEGF and nitric oxide. In addition, linoleic acid but not oleic acid induced MSC to increase production of interleukin-8. Collectively these data suggest that exposure to fatty acids may have functional consequences for MSC therapy. Fatty acids may affect MSC engraftment to injured tissue and MSC secretion of cytokines and growth factors that regulate local cellular responses to injury.

Derivation of islet‐like cells from mesenchymal stem cells using PDX1‐transducing lentiviruses

Pancreatic duodenum homeobox protein-1 (PDX1) is a master regulatory gene in pancreatic development. Reprogramming of mesenchymal stem cells (MSCs) is a promising tool for producing insulin-producing cells. In this study, lentivirus harboring PDX1 (LV-PDX1) has been used for persistence gene expression in MSCs. The objective of this study was to evaluate the potential of lentivirus to introduce the PDX1 gene into MSCs to produce insulin-secreting cells and apply it for treatment of hyperglycemia in diabetic rats. MSCs were isolated from rat bone marrow, characterized, and transduced by LV-PDX1. Significant expressions of PDX1, neurogenin3, glucagon, glucose transporter2 (Glut2), and insulin were detected by quantitative reverse transcription-polymerase chain reaction (P < 0.05). PDX1 and insulin were detected at the protein level by immunofluorescence analysis. PDX1 could trigger a gene expression cascade that involved pancreatic endocrine differentiation and also revealed the glucose sensing ability by expressing Glut2 in high-glucose medium. The insulin secretion of MSCs(PDX1+) in the high-glucose medium was 1.75-fold higher than that secreted in the low-glucose medium (P < 0.05). MSCs(PDX1+) implanted into diabetic rats could decrease the blood glucose level from 485 mg/dL to the normal level in 3 days. This study showed MSCs(PDX1+) have the potential to be used as a viable resource in cell-based gene therapy of type 1 diabetes.

T3 effect through SCR2 on chondrogenesis in vitro

T3 effect through SCR2 on chondrogenesis in vitro

A role for the primary cilium in paracrine signaling between mechanically stimulated osteocytes and mesenchymal stem cells

Bone turnover is a mechanically regulated process, coordinated in part by the network of mechanosensitive osteocytes residing within the tissue. The recruitment and bone forming activity of the mesenchymal derived osteoblast is determined by numerous factors including mechanical loading. It is therefore somewhat surprising that although mechanically regulated signaling between the coordinating osteocytes and mesenchymal stem cells (MSCs) should exist, to date it has not been directly demonstrated. In this study, conditioned media from mechanically stimulated osteocytes (MLO-Y4 cell line) was collected and added to MSCs (C3H10T1/2 cell line). The addition of mechanically stimulated osteocyte conditioned media resulted in a significant upregulation of the osteogenic genes OPN and COX-2 in MSCs compared to statically cultured conditioned media, demonstrating a novel paracrine signaling mechanism between the two cell types. The same mechanically conditioned media did not alter gene expression in osteoblasts (MC3T3 cell line), and mechanically stimulated osteoblast conditioned media did not alter gene expression in MSCs demonstrating that this signaling is unique to osteocytes and MSCs. Finally, the upregulation in osteogenic genes in MSCs was not observed if primary cilia formation was inhibited prior to mechanical stimulation of the osteocyte. In summary, the results of this study indicate that soluble factors secreted by osteocytes in response to mechanical stimulation can enhance osteogenic gene expression in MSCs demonstrating a novel, unique signaling mechanism and introduces a role for the primary cilium in flow mediated paracrine signaling in bone thereby highlighting the cilium as a potential target for therapeutics aimed at enhancing bone formation.

Wnt Signaling Promotes Neuronal Differentiation from Mesenchymal Stem Cells Through Activation of Tlx3

Wnt/β-catenin signaling promotes neural differentiation by activation of the neuron-specific transcription factors, Neurogenin1 (Ngn1), NeuroD, and Brn3a, in the nervous system. As neurons in cranial sensory ganglia and dorsal root ganglia transiently express Ngn1, NeuroD, and Brn3a during embryonic development, we hypothesized that Wnt proteins could instructively promote a sensory neuronal fate from mesenchymal stem cells (MSCs) directed to differentiate into neurons. Consistent with our hypothesis, Wnt1 induced expression of sensory neuron markers including Ngn1, NeuroD, and Brn3a, as well as glutamatergic markers in neurally induced MSCs in vitro and promoted engraftment of transplanted MSCs in the inner ear bearing selective loss of sensory neurons in vivo. Given the consensus function of T-cell leukemia 3 (Tlx3), as a glutamatergic selector gene, we postulated that the effects of canonical Wnt signaling on sensory neuron and glutamatergic marker gene expression in MSCs may be mediated by Tlx3. We first confirmed that Wnt1 indeed upregulates Tlx3 expression, which can be suppressed by canonical Wnt inhibitors. Next, our chromatin immunoprecipitation assays revealed that T-cell factor 3/4, Wnt-activated DNA binding proteins, interact with a regulatory region of Tlx3 in MSCs after neural induction. Furthermore, we demonstrated that forced expression of Tlx3 in MSCs induced sensory and glutamatergic neuron markers after neural induction. Together, these results identify Tlx3 as a novel target for canonical Wnt signaling that confers somatic stem cells with a sensory neuron phenotype upon neural induction.

Regulation of Mesenchymal Stem Cell Activity by Endothelial Cells

Emerging data suggest that mesenchymal stem cells (MSCs) are part of a periendothelial niche, suggesting the existence of heterotypic cell-cell crosstalk between endothelial cells and MSCs that regulate MSCs in their local microenvironment. We determined the effects of paracrine factors secreted by human umbilical vein endothelial cells (HUVECs) on MSC survival, proliferation, and differentiation by using an optimized, serum-free HUVEC-conditioned medium (CM). HUVEC-CM induced a significant increase in the size and number of colony-forming units-fibroblast (CFU-F) and CFU-osteoblast (CFU-O) and stimulated the proliferation of MSCs as determined by 5-bromo-2'-deoxyuridine incorporation, compared with non-CM. We also demonstrated that CM significantly enhanced the osteogenic differentiation of MSCs as shown by alkaline phosphatase enzyme histochemistry and von Kossa staining of mineralized nodules as well as by quantitative reverse transcriptase-polymerase chain reaction analysis of osteogenic markers. In contrast, there was no effect on the adipogenic differentiation of MSCs. Bioinformatic integration of HUVEC and MSC gene expression datasets identified several candidate signaling pathways responsible for mediating these effects, including fibroblast growth factor, Wnt, bone morphogenetic protein, and Notch. These data suggest strongly that endothelial cells secrete a soluble factor (or factors) that stimulates progenitor cell activity and, selectively, the osteogenic differentiation of MSCs that could contribute to niche exit.

Human mesenchymal stem cell proliferation and osteogenic differentiation during long-term ex vivo cultivation is not age dependent

Mesenchymal stem cells (MSCs) are of major clinical interest for the development of cell-based strategies to treat musculoskeletal diseases including critical-size bone defects caused by trauma, degenerative disorders, or infections. Elderly people mainly suffer from critical-size bone defects from the rising incidence of trauma, osteoporosis, and arthroplasties. In this study we investigated the influence of donor age on proliferation and osteogenic differentiation in long-term ex vivo cultures of primary human MSCs from patients in different age groups. Fifteen patients (8 men/7 women) comprised three age groups: (I) <50 years, (II) 50-65 years, and (III) >65 years. MSCs harvested from bone marrow derived from routine surgical procedures were isolated and cultured in standard medium over eight passages. Osteogenic differentiation was induced by dexamethasone (10 nM), ascorbic acid (300 μM), and β-glycerophosphate (3.5 mM). Osteogenic differentiation capacity of MSCs was quantified by alkaline phosphatase (ALP) activity, fluorescence-activated cell sorting (FACS) analysis of the surface markers CD9, CD90, CD54, CD166, CD105, CD44, and CD73, and RT-PCR for Coll I and II, Cbfa 1, ALP, OC, BSP1, and GAPDH genes characterized the phenotypic changes during monolayer expansion. In vitro chondrogenic differentiation was analyzed by immunohistochemistry and RT-PCR. Progenitor cells could be expanded in the long term from all bone marrow donations. FACS single staining analysis from MSCs showed no significant difference between the age groups. The surface antigen CD166 was predominantly found in all cell cultures independently of differentiation stage. Comparison of expanded and differentiated MSCs within a single age group showed that undifferentiated MSCs had higher CD44 levels. Osteogenic stimulation of MSCs was confirmed by measuring ALP activity. The highest ALP activity was found in probands of the age group >65 years. Additionally, we observed a tendency toward male-specific ALP increase during differentiation. Osteogenic marker gene expression in MSCs was detected by RT-PCR. No significant expression differences were detected between the three donor age groups. Micromass culture of MSCs resulted histologically and immunohistologically in a chondrogenic phenotype. Elderly osteoprogenitor cell donors are a highly clinically relevant patient population. In summary, cultivation leads to a reduced osteogenic differentiation capacity regardless of age. Because donor age does not affect osteogenic differentiation potential, it should not be used as an exclusion criterion for autologous transplantation of human adult MSCs.

Enhanced green fluorescence protein reporter gene labeling of mesenchymal stem cells mediated by lentivirus

To explore the effect of enhanced green fluorescence protein (EGFP) labeling mediated by lentivirus on the biophysical properties of mesenchymal stem cells (MSC), and whether the EGFP gene expression is permanent and stable. MSC were infected with EGFP lentivirus at different virus multiplicity of infection (MOI). EGFP positive rate was measured with fluorescent-activated cell scanning (FACS) analysis, and EGFP expression in MSC was investigated under a fluorescence microscope. Cell viability, proliferation, apoptosis and cell cycle were detected with trypan blue stain, MTT colorimetric assay, Hoechst stain and FACS analysis respectively. To evaluate the stability of EGFP expression, EGFP lentivirus infected MSC were harvested after cultured continuously in vitro for 2, 4, 8 or 16 weeks, and EGFP positive rate and fluorescence strength were detected with FACS analysis. After infected with EGFP lentivirus (MOI = 20) for 96 h, EGFP positive rate of MSC was 97.39% +/- 0.68%. Cell viability, proliferation, apoptosis and cell cycle of MSC infected with EGFP lentivirus were unaffected, as compared with control MSC (P > 0.05). When cultured in vitro continuously for 2, 4, 8 or 16 weeks, EGFP positive rates of EGFP-MSC were 97.50% +/- 0.54%, 97.32% +/- 0.51%, 97.39% +/- 0.11%, and 97.48% +/- 0.13% respectively, while EGFP fluorescence strength were 440 +/- 13, 445 +/- 12, 458 +/- 13 and 456 +/- 16 respectively. Both EGFP positive rate and fluorescence strength kept in a stable level. EGFP lentivirus can efficiently label MSC and has no significant effect on the biophysical properties of MSC. EGFP gene expression in MSC is permanent and stable. EGFP-MSC can be used for further cell tracing research.

Autocrine production of TGF-beta1 promotes myofibroblastic differentiation of neonatal lung mesenchymal stem cells.

We have isolated mesenchymal stem cells (MSCs) from tracheal aspirates of premature infants with respiratory distress. We examined the capacity of MSCs to differentiate into myofibroblasts, cells that participate in lung development, injury, and repair. Gene expression was measured by array, qPCR, immunoblot, and immunocytochemistry. Unstimulated MSCs expressed mRNAs encoding contractile (e.g., ACTA2, TAGLN), extracellular matrix (COL1A1 and ELN), and actin-binding (DBN1, PXN) proteins, consistent with a myofibroblast phenotype, although there was little translation into immunoreactive protein. Incubation in serum-free medium increased contractile protein (ACTA2, MYH11) gene expression. MSC-conditioned medium showed substantial levels of TGF-beta1, and treatment of serum-deprived cells with a type I activin receptor-like kinase inhibitor, SB-431542, attenuated the expression of genes encoding contractile and extracellular matrix proteins. Treatment of MSCs with TGF-beta1 further induced the expression of mRNAs encoding contractile (ACTA2, MYH11, TAGLN, DES) and extracellular matrix proteins (FN1, ELN, COL1A1, COL1A2), and increased the protein expression of alpha-smooth muscle actin, myosin heavy chain, and SM22. In contrast, human bone marrow-derived MSCs failed to undergo TGF-beta1-induced myofibroblastic differentiation. Finally, primary cells from tracheal aspirates behaved in an identical manner as later passage cells. We conclude that human neonatal lung MSCs demonstrate an mRNA expression pattern characteristic of myofibroblast progenitor cells. Autocrine production of TGF-beta1 further drives myofibroblastic differentiation, suggesting that, in the absence of other signals, fibrosis represents the "default program" for neonatal lung MSC gene expression. These data are consistent with the notion that MSCs play a key role in neonatal lung injury and repair.

The effect of an external magnetic force on cell adhesion and proliferation of magnetically labeled mesenchymal stem cells

BackgroundAs the strategy for tissue regeneration using mesenchymal stem cells (MSCs) for transplantation, it is necessary that MSCs be accumulated and kept in the target area. To accumulate MSCs effectively, we developed a novel technique for a magnetic targeting system with magnetically labeled MSCs and an external magnetic force. In this study, we examined the effect of an external magnetic force on magnetically labeled MSCs in terms of cell adhesion and proliferation.MethodsMagnetically labeled MSCs were plated at the bottom of an insert under the influence of an external magnetic force for 1 hour. Then the inserts were turned upside down for between 1 and 24 hours, and the number of MSCs which had fallen from the membrane was counted. The gene expression of MSCs affected magnetic force was analyzed with microarray. In the control group, the same procedure was done without the external magnetic force.ResultsAt 1 hour after the inserts were turned upside down, the average number of fallen MSCs in the magnetic group was significantly smaller than that in the control group, indicating enhanced cell adhesion. At 24 hours, the average number of fallen MSCs in the magnetic group was also significantly smaller than that in control group. In the magnetic group, integrin alpha2, alpha6, beta3 BP, intercellular adhesion molecule-2 (ICAM-2), platelet/endothelial cell adhesion molecule-1 (PECAM-1) were upregulated. At 1, 2 and 3 weeks after incubation, there was no statistical significant difference in the numbers of MSCs in the magnetic group and control group.ConclusionsThe results indicate that an external magnetic force for 1 hour enhances cell adhesion of MSCs. Moreover, there is no difference in cell proliferation after using an external magnetic force on magnetically labeled MSCs.

Derivation and characterization of human fetal MSCs: An alternative cell source for large-scale production of cardioprotective microparticles

The therapeutic effects of mesenchymal stem cells (MSCs) transplantation are increasingly thought to be mediated by MSC secretion. We have previously demonstrated that human ESC-derived MSCs (hESC-MSCs) produce cardioprotective microparticles in pig model of myocardial ischemia/reperfusion (MI/R) injury. As the safety and availability of clinical grade human ESCs remain a concern, MSCs from fetal tissue sources were evaluated as alternatives. Here we derived five MSC cultures from limb, kidney and liver tissues of three first trimester aborted fetuses and like our previously described hESC-derived MSCs; they were highly expandable and had similar telomerase activities. Each line has the potential to generate at least 1016–19 cells or 107–10 doses of cardioprotective secretion for a pig model of MI/R injury. Unlike previously described fetal MSCs, they did not express pluripotency-associated markers such as Oct4, Nanog or Tra1-60. They displayed a typical MSC surface antigen profile and differentiated into adipocytes, osteocytes and chondrocytes in vitro. Global gene expression analysis by microarray and qRT-PCR revealed a typical MSC gene expression profile that was highly correlated among the five fetal MSC cultures and with that of hESC-MSCs (r2>0.90). Like hESC-MSCs, they produced secretion that was cardioprotective in a mouse model of MI/R injury. HPLC analysis of the secretion revealed the presence of a population of microparticles with a hydrodynamic radius of 50–65 nm. This purified population of microparticles was cardioprotective at ∼1/10 dosage of the crude secretion.

Gene expression profile of mesenchymal stem cells from paired umbilical cord units: cord is different from blood.

Mesenchymal stem cells (MSC) are multipotent cells which can be obtained from several adult and fetal tissues including human umbilical cord units. We have recently shown that umbilical cord tissue (UC) is richer in MSC than umbilical cord blood (UCB) but their origin and characteristics in blood as compared to the cord remains unknown. Here we compared, for the first time, the exonic protein-coding and intronic noncoding RNA (ncRNA) expression profiles of MSC from match-paired UC and UCB samples, harvested from the same donors, processed simultaneously and under the same culture conditions. The patterns of intronic ncRNA expression in MSC from UC and UCB paired units were highly similar, indicative of their common donor origin. The respective exonic protein-coding transcript expression profiles, however, were significantly different. Hierarchical clustering based on protein-coding expression similarities grouped MSC according to their tissue location rather than original donor. Genes related to systems development, osteogenesis and immune system were expressed at higher levels in UCB, whereas genes related to cell adhesion, morphogenesis, secretion, angiogenesis and neurogenesis were more expressed in UC cells. These molecular differences verified in tissue-specific MSC gene expression may reflect functional activities influenced by distinct niches and should be considered when developing clinical protocols involving MSC from different sources. In addition, these findings reinforce our previous suggestion on the importance of banking the whole umbilical cord unit for research or future therapeutic use.Electronic supplementary materialThe online version of this article (doi:10.1007/s12015-009-9098-5) contains supplementary material, which is available to authorized users.

Indirect co-culture with tenocytes promotes proliferation and mRNA expression of tendon/ligament related genes in rat bone marrow mesenchymal stem cells

Recent evidences have suggested that humoral factors released from the appropriate co-cultured cells influenced the expansion and differentiation of mesenchymal stem cells (MSCs). However, little is known about the proliferation and differentiation of MSCs subjected to co-culture condition with tenocytes. In this study, we aimed to establish a co-culture system of MSCs and tenocytes and investigate the proliferation and tendon/ligament related gene expression of MSCs. MTT assay was used to detect the expansion of MSCs. Semi-quantitative RT-PCR was performed to investigate the expression of proliferation associated c-fos gene and tendon/ligament related genes, including type I collagen (Col I), type III collagen (Col III), tenascin C and scleraxis. Significant increase in MSCs expansion was observed after 3 days of co-culture with tenocytes. The c-fos gene expression was found distinctly higher than for control group on day 4 and day 7 of co-culture. The mRNA expression of four tendon/ligament related genes was significantly up-regulated after 14 days of co-culture with tenocytes. Thus, our research indicates that indirect co-culture with tenocytes promotes the proliferation and mRNA expression of tendon/ligament related genes in MSCs, which suggests a directed differentiation of MSCs into tendon/ligament.

Imaging Gene Expression in Human Mesenchymal Stem Cells: From Small to Large Animals

To evaluate the feasibility of reporter gene imaging in implanted human mesenchymal stem cells (MSCs) in porcine myocardium by using clinical positron emission tomography (PET)-computed tomography (CT) scanning. Animal protocols were approved by the Institutional Administrative Panel on Laboratory Animal Care. Transduction of human MSCs by using different doses of adenovirus that contained a cytomegalovirus (CMV) promoter driving the mutant herpes simplex virus type 1 thymidine kinase reporter gene (Ad-CMV-HSV1-sr39tk) was characterized in a cell culture. A total of 2.25 x 10(6) transduced (n = 5) and control nontransduced (n = 5) human MSCs were injected into the myocardium of 10 rats, and reporter gene expression in human MSCs was visualized with micro-PET by using the radiotracer 9-(4-[fluorine 18]-fluoro-3-hydroxymethylbutyl)-guanine (FHBG). Different numbers of transduced human MSCs suspended in either phosphate-buffered saline (PBS) (n = 4) or matrigel (n = 5) were injected into the myocardium of nine swine, and gene expression was visualized with a clinical PET-CT. For analysis of cell culture experiments, linear regression analyses combined with a t test were performed. To test differences in radiotracer uptake between injected and remote myocardium in both rats and swine, one-sided paired Wilcoxon tests were performed. In swine experiments, a linear regression of radiotracer uptake ratio on the number of injected transduced human MSCs was performed. In cell culture, there was a viral dose-dependent increase of gene expression and FHBG accumulation in human MSCs. Human MSC viability was 96.7% (multiplicity of infection, 250). Cardiac FHBG uptake in rats was significantly elevated (P < .0001) after human MSC injection (0.0054% injected dose [ID]/g +/- 0.0007 [standard deviation]) compared with that in the remote myocardium (0.0003% ID/g +/- 0.0001). In swine, myocardial radiotracer uptake was not elevated after injection of up to 100 x 10(6) human MSCs (PBS group). In the matrigel group, signal-to-background ratio increased to 1.87 after injection of 100 x 10(6) human MSCs and positively correlated (R(2) = 0.97, P < .001) with the number of administered human MSCs. Reporter gene imaging in human MSCs can be translated to large animals. The study highlights the importance of co-administering a "scaffold" for increasing intramyocardial retention of human MSCs.

Pressure and Distortion Regulate Human Mesenchymal Stem Cell Gene Expression

While the concept that physical forces such as tension and compression are involved in mature tissue modeling is widely accepted, the role of these specific types of mechanical loading in the differentiation and maturation of uncommitted cell types like human mesenchymal stem cells (hMSCs) is currently unknown. We observed that hMSCs have the fundamental ability to distinguish between dynamic tensile and compressive loading by regulating distinct gene expression patterns and that these differences in gene expression can be related to conformational changes in cell shape and volume. Dynamic tension was found to regulate both fibroblastic and osteogenic associated genes while dynamic compression up-regulated genes associated with chondrogenesis. Identifying genes involved in the mechanotransduction of different modes of physical loading in hMSC may greatly enhance the ability to rationally design tissue regeneration systems to restore proper tissue function.

Comparison of Cytokine Expression in Mesenchymal Stem Cells from Human Placenta, Cord Blood, and Bone Marrow

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation into lineages of mesenchymal tissues that are currently under investigation for a variety of therapeutic applications. The purpose of this study was to compare cytokine gene expression in MSCs from human placenta, cord blood (CB) and bone marrow (BM). The cytokine expression profiles of MSCs from BM, CB and placenta (amnion, decidua) were compared by proteome profiler array analysis. The cytokines that were expressed differently, in each type of MSC, were analyzed by real-time PCR. We evaluated 36 cytokines. Most types of MSCs had a common expression pattern including MIF (GIF, DER6), IL-8 (CXCL8), Serpin E1 (PAI-1), GROα(CXCL1), and IL-6. MCP-1, however, was expressed in both the MSCs from the BM and the amnion. sICAM-1 was expressed in both the amnion and decidua MSCs. SDF-1 was expressed only in the BM MSCs. Real-time PCR demonstrated the expression of the cytokines in each of the MSCs. The MSCs from bone marrow, placenta (amnion and decidua) and cord blood expressed the cytokines differently. These results suggest that cytokine induction and signal transduction are different in MSCs from different tissues.

280 MULTILINEAGE POTENTIAL OF PORCINE BONE MARROW AND ADIPOSE-DERIVED MESENCHYMAL STEM CELLS IN 3-D ALGINATE HYDROGELS

Adipose tissue presents an appealing alternative to bone marrow as a source of mesenchymal stem cells (MSC). However, in order to enhance cell proliferation and differentiation, 3-dimensional (3-D) culture may be required. A 3-D culture has benefits due to its more in vivo-like environment. Further, to form a functional tissue, a scaffold material is required to ensure proper shape and allow for efficient delivery of nutrients and growth factors. Alginate, a resorbable hydrogel, is a potential injectable scaffold for fat and bone tissue engineering due to its high biocompatibility, gelation with calcium and slow dissolution in a physiologic environment. In the present study, we examined the viability, gene expression and morphology of MSC, isolated from porcine adipose (ADSC) and bone marrow (BMSC), during osteogenic and adipogenic differentiation in a 3D alginate hydrogel environment for 0, 7 and 14 days (d). ADSC and BMSC were infused into alginate hydrogels, which polymerized upon the addition of Ca+2 ions. Both stem cell types were differentiated into osteoblasts using 0.1 μm dexamethasone, 10 mm beta glycerophosphate and 50 μm ascorbic acid, whereas adipocytes were differentiated using 10 μm insulin, 1 μm dexamethasone, and 0.5 mm IBMX. Osteogenic differentiation was confirmed using alkaline phosphatase, Von Kossa, and alizarin red S staining and adipogenic differentiation was confirmed using Oil Red O. Cell viability and proliferation was quantified using the MTT assay. Gene expression was measured using qPCR. The morphology of ADSC and BMSC differentiated toward osteogenic lineages changed with both cell types forming osteogenic nodules over time. The nodules formed by ADSC were larger in diameter than those formed by BMSC. Unlike the osteogenic cells that formed nodules, the ADSC and BMSC differentiated into adipogenic cells showed no significant changes in cell size or aggregation. Gene expression results indicated increased PPARG expression in BMSC with time whereas ADSC showed a peak of expression on day 7 and then decreased. ADSC showed increased (14-fold) PPRG expression when compared with BMSC. ADSC had 160-fold less expression of ALP than BMSC. BMSC showed a 16-fold higher expression level of BGLAP than ADSC. ADSC showed a 15.8% higher expression than BMSC for COL1a1. Both ADSC and BMSC showed similar trends SPARC expression, but BMSC had a 12-fold higher expression of SPP1 than ADSC. In summary, both types of mesenchymal stem cells successfully differentiated into both lineages and maintained viability in the hydrogel over time. In conclusion, alginate is a viable scaffold material for the differentiation of mesenchymal stem cells for tissue engineering applications. These results allow for future studies using the pig as an in vivo fat and bone tissue engineering model. This research was supported by the Illinois Regenerative Medicine Institute.

Regulation of P450 oxidoreductase by gonadotropins in rat ovary and its effect on estrogen production

BackgroundP450 oxidoreductase (POR) catalyzes electron transfer to microsomal P450 enzymes. Its deficiency causes Antley-Bixler syndrome (ABS), and about half the patients with ABS have ambiguous genitalia and/or impaired steroidogenesis. POR mRNA expression is up-regulated when mesenchymal stem cells (MSCs) differentiate into steroidogenic cells, suggesting that the regulation of POR gene expression is important for steroidogenesis. In this context we examined the regulation of POR expression in ovarian granulosa cells by gonadotropins, and its possible role in steroidogenesis.MethodsChanges in gene expression in MSCs during differentiation into steroidogenic cells were examined by DNA microarray analysis. Changes in mRNA and protein expression of POR in the rat ovary or in granulosa cells induced by gonadotropin treatment were examined by reverse transcription-polymerase chain reaction and western blotting. Effects of transient expression of wild-type or mutant (R457H or V492E) POR proteins on the production of estrone in COS-7 cells were examined in vitro. Effects of POR knockdown were also examined in estrogen producing cell-line, KGN cells.ResultsPOR mRNA was induced in MSCs following transduction with the SF-1 retrovirus, and was further increased by cAMP treatment. Expression of POR mRNA, as well as Cyp19 mRNA, in the rat ovary were induced by equine chorionic gonadotropin and human chorionic gonadotropin. POR mRNA and protein were also induced by follicle stimulating hormone in primary cultured rat granulosa cells, and the induction pattern was similar to that for aromatase. Transient expression of POR in COS-7 cells, which expressed a constant amount of aromatase protein, greatly increased the rate of conversion of androstenedione to estrone, in a dose-dependent manner. The expression of mutant POR proteins (R457H or V492E), such as those found in ABS patients, had much less effect on aromatase activity than expression of wild-type POR proteins. Knockdown of endogenous POR protein in KGN human granulosa cells led to reduced estrone production, indicating that endogenous POR affected aromatase activity.ConclusionWe demonstrated that the expression of POR, together with that of aromatase, was regulated by gonadotropins, and that its induction could up-regulate aromatase activity in the ovary, resulting in a coordinated increase in estrogen production.