MSC Cells Research Articles

Abstract 018: Sox6 is a Critical Factor in Renin Cell Fate in Development and Pathology

Introduction: the renin-angiotensin system (RAS) is an important component of blood pressure regulation in mammals. Renin, primarily expressed and secreted by kidney Juxtaglomerular (JG) cells, catalyzes the rate limiting step in RAS. However, the transcriptional mechanisms that govern the specification of renin expressing cells in development and under normal or pathophysiological conditions remain poorly understood. We sought to determine new regulators of renin cell fate during kidney development and JG recruitment. Methods: Gene expression profiles of renal MSC and JG cells were determined by Affymetrix Mouse 430 2.0 array. JG cells and renal MSC were isolated from adult C57Bl6 Ren1c YFP mice by collagenase digestion of the kidney, followed by flow cytometry to select for the cells. Renin expression in vitro was induced by treatment with IBMX and Forskolin. Renal MSCs were transduced with lentivirus carrying vectors for Sox6, Sox6 shRNA or controls. Ex vivo analysis was performed in embryonic kidneys (14.5 dpc) isolated and transduced with Sox6 shRNA or scrambled shRNA. The kidneys were then cultured for 4 days. Results: Microarray data showed that the transcription factor Sox6 was highly expressed in FACS isolated JG cells compared to renal MSC (96-fold, n=3, P<0.05). This finding was validated by qPCR (100-fold, n=4, P<0.05). Sox6 was expressed in renin producing cells during kidney embryogenesis as determined by immunofluorescence and confocal microscopy (E14.5, N=4). Knockdown of Sox6 by shRNA in an ex vivo model of kidney development resulted in a 70% reduction of renin expression (N=4, P<0.01). Differentiation of adult renal MSCs to renin producing cells in vitro was enhanced by overexpression of Sox6 (20-fold, N=6, P<0.01). Knockdown of Sox6 by shRNA inhibited in vitro MSC differentiation (5-fold, N=6, P<0.001). In vivo, low salt and furosemide, which stimulates JG recruitment, increased Sox6 expression (5-fold, N=5, P<0.001) and colocalization with renin. Conclusion: Our results support a novel and critical role for the transcription factor Sox6 in renin cell fate and in renal development and physiology. Further studies can provide an in-depth understanding of the role of Sox 6 in hypertension and its therapy.

Mesenchymal stem cell-based HSP70 promoter-driven VEGFA induction by resveratrol promotes angiogenesis in a mouse model.

Several studies of stem cell-based gene therapy have indicated that long-lasting regeneration following vessel ischemia may be stimulated through VEGFA gene therapy and/or MSC transplantation for reduction of ischemic injury in limb ischemia and heart failure. The therapeutic potential of MSC transplantation can be further improved by genetically modifying MSCs with genes which enhance angiogenesis following ischemic injury. In the present study, we aimed to develop an approach in MSC-based therapy for repair and mitigation of ischemic injury and regeneration of damaged tissues in ischemic disease. HSP70 promoter-driven VEGFA expression was induced by resveratrol (RSV) in MSCs, and in combination with known RSV biological functions, the protective effects of our approach were investigated by using ex vivo aortic ring coculture system and a 3D scaffolds in vivo model. Results of this investigation demonstrated that HSP promoter-driven VEGFA expression in MSC increased approximately 2-fold over the background VEGFA levels upon HSP70 promoter induction by RSV. Exposure of HUVEC cells to medium containing MSC in which VEGFA had been induced by cis-RSV enhanced tube formation in the treated HUVEC cells. RSV-treated MSC cells differentiated into endothelial-like phenotypes, exhibiting markedly elevated expression of endothelial cell markers. These MSCs also induced aortic ring sprouting, characteristic of neovascular formation from pre-existing vessels, and additionally promoted neovascularization at the MSC transplantation site in a mouse model. These observations support a hypothesis that VEGFA expression induced by cis-RSV acting on the HSP70 promoter in transplanted MSC augments the angiogenic effects of stem cell gene therapy. The use of an inducible system also vastly reduces possible clinical risks associated with constitutive VEGFA expression.

Membrane-type 1 matrix metalloproteinase controls osteo- and chondrogenesis by a proteolysis-independent mechanism mediated by its cytoplasmic tail

s / Osteoarthritis and Cartilage 23 (2015) A26eA81 A64 deregulated genes included Has1, ADAMTS4, Tnf, IL6, IL18, Inhba, Cd68, Ngf, Ccr2, Wnt 16, Tnfaip6 and Il1r. Conclusions: Deletion of JNK2 retarded the development of experimental murine OA, showing the importance of the kinase for cartilage homeostasis in vivo and implicating an intracellular signalling pathway in OA for the first time. 68 MEMBRANE-TYPE 1 MATRIX METALLOPROTEINASE CONTROLS OSTEOAND CHONDROGENESIS BY A PROTEOLYSIS-INDEPENDENT MECHANISM MEDIATED BY ITS CYTOPLASMIC TAIL Q. Yang y, M. Attur y, T. Kirsch z, Y.J. Lee z, S. Yakar x, Z. Liu x, S.B. Abramson y, P. Mignatti yk. yNYU Sch. of Med. Dept. of Med., New York, NY, USA; zNYU Sch. of Med. Dept. of Orthopedic Surgery, New York, NY, USA; xNYU Coll. of Dentistry Dept. of Basic Sci. and Craniofacial Biology, New York, NY, USA; kNYU Sch. of Med. Dept. of Cell Biology, New York, NY, USA Purpose: We aimed to understand the mechanism by which membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14) controls bone and cartilage homeostasis. MT1-MMP, a cell-membrane-bound proteinase with an extracellular catalytic site and a 20-amino acid cytoplasmic tail, degrades the extracellular matrix and plays a key role in postnatal bone formation. MT1-MMP is overexpressed in osteoarthritis, and mutation of MT1-MMP in humans causes the multicentric osteolysis and arthritis disease, Winchester syndrome. The genetic deficiency of MT1-MMP (MT1-MMP-/-) in the mouse causes dwarfism, osteopenia and severe arthritis. Conditional deletion of MT1-MMP in bone marrow-derived mesenchymal progenitor cells (BM-MSC) fully recapitulates this phenotype, showing that MT1-MMP controls osteogenic differentiation inMSC. It has been proposed that the phenotype of MT1-MMP-/mice results from the lack of MT1-MMP proteolytic activity. However, mounting evidence shows a variety of proteolysisindependent functions of MT1-MMP. The unique tyrosine (Y573) in the MT1-MMP cytoplasmic tail is fundamental for the control of intracellular signaling. We have shown that Y573 mediates Ras-ERK1/2 signaling, and that Y573 mutation (Y573D) blocks this functionwithout affecting MT1-MMP proteolytic activity. Methods: We generated a mouse with the Y573D mutation in MT1MMP (MT1-MMP Y573D) and characterized its skeletal phenotype by histological and microCT analyses. We isolated BM-MSC and induced them to differentiate into osteoblasts, chondrocytes and adipocytes, using qRT-PCR to analyze gene expression. Mouse C3H10T1/2MSCwere transfected with MT1-MMP cDNA and analyzed for Wnt signaling and Runx2 activation by luciferase reporter assays. Results: Analysis of the long bones of both homozygote and heterozygote MT1-MMP Y573D mice showed increased trabecular bone relative to wt littermates (BV/TV %; wt: 10 4±8; homo: 23.2±2.0; Tb.Th (mm) wt: 20.5±3.8; homo: 40.1±7.0; Tb.N./mm wt: 10.5±4.2; homo: 19.5 4.2; Tb.Sp (mm): 325.2±52.1; 190.6 49.1; p 0.01), a phenotypeopposite to that of MT1-MMP-/mice. MT1-MMP Y573D mice also showed marked thinning of articular cartilage, with disorganized tissue architecture, clustering and cloning of chondrocytes, as well as pronounced decrease in bone marrow-associated and total body fat (fat weight/body weight wt: 0.270±0.0105; homo: 0.0135±0.006; p1⁄40.0029). These findings indicated a defect in MSC differentiation. Therefore, we isolated BM-MSC from wt and homozygous mutant littermates, and induced them to differentiate into osteoblast and chondrocytes, and myeloid precursors were induced to differentiate into osteoclasts. The Y573D mutation dramatically increased MSC expression of osteoblast markers (ALP, COL1, BSP, OCN, RUNX2) and strongly downregulated chondrocyte (ADA4, ADA5, SOX9, MMP13) and osteoclast markers (TRACP, CALCR, CTSK, DC-STAMP, MMP9, iNOS). These findings indicated thatWnt signaling is upregulated inMT1MMP Y573D-expressing MSC. The canonical Wnt pathway is indeed unique, as it exerts opposite effects on osteoblast and chondrocyte differentiation fromMSC. Therefore, we analyzedWnt signaling and Runx2 activation. We transiently transfected C3H10T1/2 MSC cells in osteoblast medium with the cDNAs for wt MT1-MMP and MT1-MMP Y573D. As controls the cellswere transfectedwith the empty vector (pcDNA) orwith MT1-MMP E240A, a mutant devoid of proteolytic activity. MT1-MMP Y573D dramatically upregulated both Wnt signaling (2.8-4.6-fold; p1⁄40.007) and Runx2 activity (8.7-46.5-fold; 1⁄40.009) relative to wt MT1MMP and MT1-MMP E240A. Conclusions: MT1-MMP controls Wnt signaling by a mechanism independent of extracellular proteolysis and mediated by its cytoplasmic tail. MT1-MMP is a bifunctional protein, with an extracellular proteolytic activity that promotes bone formation through ECM remodeling and a cytoplasmic tail that controls osteogenesis by interacting with a key pro-osteogenic signaling pathway. 69 A DUAL ROLE FOR NOTCH SIGNALING IN JOINT CARTILAGE MAINTENANCE AND OSTEOARTHRITIS Z. Liu y, J. Chen z, A.J. Mirando z, C. Wang y, M.J. Zuscik y, R.J. O'Keefe x, M.J. Hilton z. yUniv. of Rochester Med. Ctr., Rochester, NY, USA; zDuke Univ. Sch. of Med., Durham, NC, USA; xWashington Univ. Sch. of Med.,

MSCs: Scientific Support for Multiple Therapies.

MSCs: Scientific Support for Multiple Therapies.

In Vitro Tissue Regeneration by Human Degenerated Nucleus Pulposus Cells in Hyperosmotic Culture Medium

Introduction Healthy intervertebral disc (IVD) cells normally reside in a high osmolality environment of 450 to 550 mOsm/kg and extracellular matrix production by MSC cells has been shown to increase with increasing osmolality. However, it is not known how human degenerated disc cells are affected by osmolality. The aim of the current study was to determine the effects of osmolality on tissue regeneration by human degenerated NP cells. Materials and Methods The osmolality of standard chondrogenic culture medium (DMEM, 2% ascorbic acid-2 phosphate, 2% human serum albumin, 2% insulin-transferring selenium-X, 1% penicillin/streptomycin, 10 ng/mL transforming growth factor β2 [TGF-β2]) was adjusted with NaCl and sucrose from 340 to 400, 450, and 500 mOsm/kg. IVD cells from four human donors (Thompson grade III) were cultured for 28 days in high density (1*10^6 cells/cm2) on collagen II coated filters. LDH analysis was performed to analyze toxicity. Samples were stained with Safranin-O for glycosaminoglycans (GAGs) and with immunohistochemistry for collagen II. Gene expression was evaluated by qPCR and a collagen low-density array. Matrix content as reflected by glycosaminoglycan (GAG) production was measured with a dimethylmethylene blue (DMMB) assay and DNA content with a Picogreen assay. Statistical analysis included univariate analysis of variance with randomized block design and post hoc Dunnet t-test. Results No cytotoxic effects were found in any of the conditions tested. Gene expression showed an osmolality-dependent increase compared with standard medium in collagen II and aggrecan only when NaCl was used (see Fig. 1A, B for p-values). However, at the protein level, no effect of increased osmolality using NaCl was found. This could not be explained by MMP gene expression levels, as these seemed lower at high osmolality. Tissues cultured in the presence of the osmolyte sucrose appeared to contain less proteoglycans, but this difference was not significant. Conclusion In conclusion, in the current culture system, the increased expression of matrix genes with osmolality appears to be osmolyte dependent rather than osmolality dependent. Moreover, the absence of an effect at the protein level suggests adjusting osmolality with NaCl to enhance matrix production by IVD cells is not required. Disclosure of Interest None declared

Oncogenic transformation tunes the cross-talk between mesenchymal stem cells and T lymphocytes

Stem cells from mesenchymal origin (MSC) exert a plethora of immunomodulatory effects. We created a neoplastic model based on in vitro step-wise transformation to assess whether oncogenic pathways have the capacity to mould the cross-talk of MSC and lymphocytes. Neoplastic MSC exhibit an increased inhibitory effect on T cell proliferation, either directly or mediated by myeloid derived suppressor cells. Additionally, transformation of MSC enhances T cell apoptosis without reducing either the percentage of CD25 expressing cells or the level of this protein expression. Malignant transformation drives MSC to lose dependency on nitric oxide for immunosuppression whilst increasing the constitutive production of PGE2. Our results indicate that oncogenesis tunes the interplay between MSC and immune cells, favoring cancer immune evasion.

Cytokine milieu in the lymphatic fluid and serum of chronic pathology patients with bancroftian lymphatic filariasis.

number of apoptotic cells (255.00 105.90 vs. 121.40 81.38, p1⁄4n.s.). Coculture with MSCs did not affect apoptosis, but a reduced proliferation of both CD3+ cells and Tregs was observed. Addition of MSC-MPs to PBMC cultures on days 0 and 1 did not affect proliferation of CD3+ cells (10153 4799 vs. 6410 4175, p1⁄4n.s.), but induced a 3-fold increase of apoptosis in this cell population (826.20 549 vs. 255.00 105.90, p<0.05). Moreover, addition of MPs induced a 2-fold increase of Treg proliferation (3018.00 933.90, p<0.05). Similar effects were observed with MSCs and MSC-MPs isolated from umbilical cord. The activity of indoleamine 2,3dioxygenase (IDO), an established mediator of MSC immunosuppressive effects, was increased in supernatants of PBMCs co-cultured with MSCs, but was not affected by the presence of MSC-MPs. In conclusion, MSC-MPs demonstrate immunomodulatory effects on T cells in vitro. However, at variance with previous observations on B cells, both these effects and the underlying mechanisms appear to be different from those exhibited by the parent MSC cells.

TLR Ligands Stimulation Protects MSC from NK Killing

Mesenchymal stem cells (MSCs) play a fundamental role in allograft rejection and graft-versus-host disease through their immunosuppressive abilities. Recently, Toll-like receptors (TLR) have been shown to modulate MSC functions. The aim of this study was to investigate the effects of several TLR ligands on the interaction between MSC and natural killer (NK) cells. Our results show that TLR-primed adult bone marrow and embryonic MSC are more resistant than unprimed MSC to IL-2-activated NK-induced killing. Such protection can be explained by the modulation of Natural Killer group 2D ligands major histocompatibility complex class I chain A and ULBP3 and DNAM-1 ligands by TLR-primed MSC. These results indicate that MSCs are able to adapt their immuno-behavior in an inflammatory context, decreasing their susceptibility to NK killing. In addition, TLR3 but not TLR4-primed MSC enhance their suppressive functions against NK cells. However, the efficiency of this response is heterogeneous, even if the phenotypes of different analyzed MSC are rather homogeneous. The consequences could be important in MSC-mediated cell therapy, since the heterogeneity of adult MSC responders may be explored in order to select the more efficient responders.

From bedside to bench and back again—molecular mechanisms in acute liver failure

From bedside to bench and back again—molecular mechanisms in acute liver failure

Phosphorylation Of GSK3β Is Associated With Inferior Survival In Acute Myeloid Leukemia and Is An Indicator Of AKT Activation In AML Blasts and Bone Marrow Mesenchymal Stem Cells

▪Glycogen Synthase Kinase 3β (GSK3β) is a key regulator of cell metabolism, proliferation, survival, and differentiation. The kinase has abundant substrates including many proteins in the canonical WNT pathway. Considering that GSK3β phosphorylation of many pro-survival proteins result in their degradation (e.g MYC, MCL-1), it is not surprising that GSK3β activation by stress challenge leads to cell cycle arrest and/or apoptosis. GSK3β is negatively regulated by serine 9 phosphorylation mediated by Protein Kinase B (AKT). Since AKT activation supports survival of AML cells and inactivation of GSK3β could suppress stress signaling events, we hypothesize that serine 9 phosphorylation of GSK3β (p-GSK3β ) will be detrimental for AML patients. In the current study, we analyzed GSK3β expression by Reverse Phase Protein Analysis (RPPA) in a cohort of 511 AML patients. GSK3β expression was correlated with patient survival data and disease characteristics such as French-American-British (FAB) classification, cytogenetics, and mutational status. High levels of p-GSK3β were found to correlate with adverse outcome for survival and complete remission duration (CR) in patients with intermediate cytogenetics but not in those with unfavorable cytogenetics. CR was only 45 weeks in the third of patients with highest p-GSK3β levels compared to 98 weeks for patients with low levels (p = 0.008; N = 121). Even intermediate cytogenetic patients with FLT3 mutation fared better when p-GSK3β levels were lower (50 versus 24 weeks; p = 0.009; N = 35). Expression of GSK3β and its phosphorylated form was compared with expression of 229 other proteins using RPPA in the AML patient cohort. Consistent with p-GSK3β as an indicator of AKT activation, RPPA revealed that p-GSK3β is positively correlated with phosphorylation of AKT (S473), BAD (S136), and P70S6K. In addition, p-GSK3β negatively correlated with FOXO3A (which is degraded after phosphorylation by AKT). Bone marrow mesenchymal stem cells (BM MSCs) are a critical component of the leukemic microenvironment but how these cells modulate the survival of leukemia cells is not clear. RPPA analysis was performed on BM MSC from healthy donor (N = 71) and BM MSCs from AML patients (N = 106). Interestingly, both total and phosphorylated GSK3 were found to be elevated in the AML samples suggesting that AKT is also activated in the leukemic MSCs. In vitro models of the BM microenvironment suggest that the AKT pathway is activated in both the leukemic and supporting stromal cells so this finding is consistent with these models (Konopleva and Andreeff, Curr Drug Targets. 2007; 8: 685). Unlike in AML blast cells, FOXO3A was not correlated with GSK3β phosphorylation in the MSCs. Examination of miRs in normal versus AML BM MSCs using microarray analysis and validated by qRT-PCR indicate that miR-21 is elevated in the MSC of the normal individuals. As miR-21 is suppressed by FOXO3A, this finding supports the notion that AKT is active in the AML BM MSCs but FOXO3A may not be functional. This possibility is plausible as induction of FOXO3A results in apoptosis in MSC (Djouad Cloning Stem Cells. 2009; 11:407).ConclusionThese findings suggest that AKT mediated phosphorylation of GSK3β may be detrimental to AML patients and p-GSK3β may serve as an important prognostic factor for at least a subset of AML patients. The results also suggest that activation of AKT can occur in both the malignant cells and MSC cells in the leukemic microenvironment. Disclosures:No relevant conflicts of interest to declare.

Anti-Inflammatory Effects of Adult Stem Cells in Sustained Lung Injury: A Comparative Study

Lung diseases are a major cause of global morbidity and mortality that are treated with limited efficacy. Recently stem cell therapies have been shown to effectively treat animal models of lung disease. However, there are limitations to the translation of these cell therapies to clinical disease. Studies have shown that delayed treatment of animal models does not improve outcomes and that the models do not reflect the repeated injury that is present in most lung diseases. We tested the efficacy of amnion mesenchymal stem cells (AM-MSC), bone marrow MSC (BM-MSC) and human amniotic epithelial cells (hAEC) in C57BL/6 mice using a repeat dose bleomycin-induced model of lung injury that better reflects the repeat injury seen in lung diseases. The dual bleomycin dose led to significantly higher levels of inflammation and fibrosis in the mouse lung compared to a single bleomycin dose. Intravenously infused stem cells were present in the lung in similar numbers at days 7 and 21 post cell injection. In addition, stem cell injection resulted in a significant decrease in inflammatory cell infiltrate and a reduction in IL-1 (AM-MSC), IL-6 (AM-MSC, BM-MSC, hAEC) and TNF-α (AM-MSC). The only trophic factor tested that increased following stem cell injection was IL-1RA (AM-MSC). IL-1RA levels may be modulated by GM-CSF produced by AM-MSC. Furthermore, only AM-MSC reduced collagen deposition and increased MMP-9 activity in the lung although there was a reduction of the pro-fibrogenic cytokine TGF-β following BM-MSC, AM-MSC and hAEC treatment. Therefore, AM-MSC may be more effective in reducing injury following delayed injection in the setting of repeated lung injury.

Cells behaviors and genotoxicity on topological surface

To investigate different cells behaviors and genotoxicity, which were driven by specific microenvironments, three patterned surfaces (pillars, wide grooves and narrow grooves) and one smooth surface were prepared by template-based technique. Vinculin is a membrane-cytoskeletal protein in focal adhesion plaques and associates with cell–cell and cell–matrix junctions, which can promote cell adhesion and spreading. The immunofluorescence staining of vinculin revealed that the narrow grooves patterned substrate was favorable for L929 cell adhesion. For cell multiplication, the narrow grooves surface was fitted for the proliferation of L929, L02 and MSC cells, the pillars surface was only in favor of L929 cells to proliferate during 7days of cell cultivation. Cell genetic toxicity was evaluated by cellular micronuclei test (MNT). The results indicated that topological surfaces were more suitable for L929 cells to proliferate and maintain the stability of genome. On the contrary, the narrow grooves surface induced higher micronuclei ratio of L02 and MSC cells than other surfaces. With the comprehensive results of cell multiplication and MNT, it was concluded that the wide grooves surface was best fitted for L02 cells to proliferate and have less DNA damages, and the smooth surface was optimum for the research of MSC cells in vitro.

Simultaneous inhibition of cell-cycle, proliferation, survival, metastatic pathways and induction of apoptosis in breast cancer cells by a phytochemical super-cocktail: genes that underpin its mode of action.

Traditional chemotherapy and radiotherapy for cancer treatment face serious challenges such as drug resistance and toxic side effects. Complementary / Alternative medicine is increasingly being practiced worldwide due to its safety beneficial therapeutic effects. We hypothesized that a super combination (SC) of known phytochemicals used at bioavailable levels could induce 100% killing of breast cancer (BC) cells without toxic effects on normal cells and that microarray analysis would identify potential genes for targeted therapy of BC. Mesenchymal Stems cells (MSC, control) and two BC cell lines were treated with six well established pro-apoptotic phytochemicals individually and in combination (super cocktail), at bioavailable levels. The compounds were ineffective individually. In combination, they significantly suppressed BC cell proliferation (>80%), inhibited migration and invasion, caused cell cycle arrest and induced apoptosis resulting in 100% cell death. However, there were no deleterious effects on MSC cells used as control. Furthermore, the SC down-regulated the expression of PCNA, Rb, CDK4, BcL-2, SVV, and CD44 (metastasis inducing stem cell factor) in the BC cell lines. Microarray analysis revealed several differentially expressed key genes (PCNA, Rb, CDK4, Bcl-2, SVV, P53 and CD44) underpinning SC-promoted BC cell death and motility. Four unique genes were highly up-regulated (ARC, GADD45B, MYLIP and CDKN1C). This investigation indicates the potential for development of a highly effective phytochemical combination for breast cancer chemoprevention / chemotherapy. The novel over-expressed genes hold the potential for development as markers to follow efficacy of therapy.

Induced Pluripotent Mesenchymal Stromal Cell Clones Retain Donor-derived Differences in DNA Methylation Profiles

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is an epigenetic phenomenon. It has been suggested that iPSC retain some tissue-specific memory whereas little is known about interindividual epigenetic variation. We have reprogrammed mesenchymal stromal cells from human bone marrow (iP-MSC) and compared their DNA methylation profiles with initial MSC and embryonic stem cells (ESCs) using high-density DNA methylation arrays covering more than 450,000 CpG sites. Overall, DNA methylation patterns of iP-MSC and ESC were similar whereas some CpG sites revealed highly significant differences, which were not related to parental MSC. Furthermore, hypermethylation in iP-MSC versus ESC occurred preferentially outside of CpG islands and was enriched in genes involved in epidermal differentiation indicating that these differences are not due to random de novo methylation. Subsequently, we searched for CpG sites with donor-specific variation. These "epigenetic fingerprints" were highly enriched in non-promoter regions and outside of CpG islands-and they were maintained upon reprogramming. In conclusion, iP-MSC clones revealed relatively little intraindividual variation but they maintained donor-derived epigenetic differences. In the absence of isogenic controls, it would therefore be more appropriate to compare iPSC from different donors rather than a high number of different clones from the same patient.

Demethylation of Epiregulin Gene by Histone Demethylase FBXL11 and BCL6 Corepressor Inhibits Osteo/dentinogenic Differentiation

Mesenchymal stem cells (MSCs) are a reliable resource for tissue regeneration, but the molecular mechanism underlying directed differentiation remains unclear; this has restricted potential MSC applications. Histone methylation, controlled by histone methyltransferases and demethylases, may play a key role in MSC differentiation. Here, we investigated FBXL11, a histone demethylase, lysine (K)-specific demethylase 2A, which is evolutionarily conserved, ubiquitously expressed, and a member of the JmjC-domain-containing histone demethylase family. We tested whether FBXL11 could inhibit the osteo/dentinogenic differentiation potential in MSC cells with gain- and loss-of-function assays. We found that FBXL11 regulated osteo/dentinogenic differentiation in MSC cells. Furthermore, we found that the gene encoding the epidermal growth factor, Epiregulin (EREG), was a downstream target of FBXL11, and that EREG mediated FBXL11 regulation of MSC differentiation. Moreover, we found that the FBXL11 histone demethylase function was activated by associating with BCL6 corepressor, and this complex could repress EREG transcription by increasing histone K4/36 methylation in the EREG promoter. In conclusion, our results elucidated a new function for FBXL11 and EREG, explored the molecular mechanism underlying directed differentiation in MSC cells, and identified potential target genes for improving tissue regeneration techniques.

Alginate-Graft-PEI as a Gene Delivery Vector with High Efficiency and Low Cytotoxicity

To overcome the efficiency–cytotoxicity dilemma of native PEI and incorporate the advantages of alginate, we designed a novel gene vector by grafting PEI 2000 onto alginate, an anionic polysaccharide with excellent biocompatibility. The alginate-graft-PEI (Alg-g-PEI) was successfully synthesized and then characterized by elemental analysis, 1H-NMR and 13C-NMR. The M w of Alg-g-PEI is ca. 17 000. Acid–base titration confirmed that Alg-g-PEI retained the buffering capacity of native PEI. The DNA binding ability of the polymer was confirmed by gel retardation assay. DSL analysis showed that Alg-g-PEI had a particle size and zeta-potential similar to PEI 25K. AFM detected a clear and well-shaped morphology of the complexes. Additionally, Alg-g-PEI exhibited lower cytotoxicity than PEI 25K in BEL7402, MSC and RVMSC cells. Compared with PEI 25K, Alg-g-PEI had comparable or even higher transfection efficiency. Similarly, Alg-g-PEI-mediated VEGF expression was significantly higher compared with PEI 25K-mediated VEGF expression. All together, our results suggest that Alg-g-PEI has a potential to be a safe and efficient agent for gene therapy.

Secreted Frizzled-Related Protein-3 (sFRP3) Is Produced by Myeloma Cells and Augments Wnt3a-Induced Differentiation of Mesenchymal Stem Cells and OPG Production in Osteoblasts

Abstract 808 Background:Bone disease is one of the most debilitating complications in patients with multiple myeloma (MM). The molecular mechanisms by which MM triggers bone disease are not fully understood. We have previously demonstrated that Dkk1 is highly expressed in primary MM plasma cells, and associated with bone disease in MM patients by inhibiting Wnt signaling-promoted mesenchymal stem cell differentiation and osteoprotegerin production in osteoblast cells. We have also reported that increase in Wnt signaling in the bone marrow microenvironment by overexpression of Wnt3a in myeloma cells or administration of rWnt3a, or indirectly increasing Wnt signaling by administration of anti-Dkk1 neutralizing antibody also decreased in osteoclast numbers. However, Dkk1 is less frequently expressed in MM cell lines that are derived mostly from late stage of MM; and injection of these MM cell lines into human fetal bone also is able to induce bone lesion in MM animal model. These results indicate that additional factors may be involved in induction of the bone disease at the stage of the disease. The members of the sFRPs family of secreted proteins (including sFRP-1, -2, -3 and -4) directly bind to Wnts, thereby preventing Wnts from binding to the cellular Wnt receptor complex. It has also been reported that sFRP-1 and -2 augment canonical Wnt3a activated signaling in fibroblast. MM cells from pateints with advanced bone lesions express sFRP2 mRNA. Like sFRP2, sFRP3 mRNA is highly expressed in MM plasma cells, but it's function in MM bone disease remains unknown. We sought to investigate the role of sFRP3 in MM-triggered bone lesions using the osteoblast (OB) cell lines CH3T1/2 and C2C12, and serum from MM pateints those MM cells expressed high level of sFRP3. Methods/Results:RT-PCR analysis showed that sFRP3 is expressed in primary MM plasma cells and certain MM cell lines. Recombinant sFRP3 protein did not inhibit, but synergized with Wnt3a to increase beta-catenin protein, while Dkk1 significantly inhibited this process. Similarly, sFRP3 treatment of OB cells increase Wnt-3a-induced TCF transcript activity in OB cells transfected with TOPflash luciferase report constructs. sFRP3 also increased MSC differentiation, as evidenced by increase in alkaline phosphatase activity (ALP) and increased in mineralization by Alizarin red staining. sFRP3 treatment also increases OPG mRNA and protein production in these cells. Similar to sFRP3, sFRP1 and sFRP2 synergistically acted with Wnt3a to induce MSC differentiation and OPG expression in osteoblasts, while Dkk1 significantly inhibited these processes. To confirm the synergistic effects of sFRPs with canonical Wnt signaling on MSC differentiation, we employed R-podin1, a well-known agonist of canonical Wnt signaling. Treatments of MSC cells with R-podin1 led to increase in beta-catenin protein and TCF transcriptional activity and in ALP activity, and increase in OPG mRNA and protein. Pretreatment of the cells with sFRP2 and sFPP3 proteins further enhanced the function of R-podin1. In contrast, Dkk1 protein showed negative effect on R-Spodin1 functions, indicating that sFRP2 and sFRP3 synergized with R-Spodin1 to induce activation of canonical Wnt signaling and subsequent MSC differentiation and OPG production. Conclusion:Taken together, these data suggest that sFRP2 and sFRP3 augment canonical Wnt signaling to induce MSC differentiation and indirectly inhibit osteoclastogenesis by regulating OPG in MSC cells. These results also indicate that Dkk1 may be most important in MM-induced bone disease. Disclosures:Barlogie:Celgene, Genzyme, Novartis, Millennium: Consultancy, Honoraria, Patents & Royalties. Shaughnessy:Myeloma Health, Celgene, Genzyme, Novartis: Consultancy, Employment, Equity Ownership, Honoraria, Patents & Royalties.

Alternate PAX3 and PAX7 C-terminal isoforms in myogenic differentiation and sarcomagenesis

Pax3 and Pax7 are closely related genes that are involved in commitment of cells to a myogenic lineage during skeletal muscle development and regeneration. Several Pax3 and Pax7 transcripts are expressed from the genes, generating different isoforms with potentially distinct DNA binding and transactivation properties. The aim of this study was to investigate the implication of Pax3 and Pax7 C-terminal isoforms during myogenic differentiation and tumorigenesis, since fusions involving these genes are commonly associated with alveolar rhabdomyosarcoma (ARMS). Uncommitted (mouse mesenchymal stem cells, MSCs) and committed (C2C12) myogenic precursor cells were stably transfected with PAX3/FKHR and PAXC7/ FKHR fusion genes. We analysed gene and protein expression comparing the newly generated cells with the parental cells, to determine the functional importance of Pax3 and Pax7 C-terminal isoforms. We found that the transcript Pax3c was expressed at low levels in undifferentiated C2C12 and MSCs cells, but its expression levels increased considerably at later stages of differentiation. However, expression levels of Pax3d transcript increased only slightly after differentiation. Pax7 transcripts, present before differentiation in committed C2C12 cells, but absent in uncommitted MSCs, increased noticeably in MSCs after differentiation. We also found that the presence of PAX/FKHR fusions prevented both C2C12 and MSC cells from terminal myogenic differentiation and increased the expression of discrete endogenous Pax3/7 transcripts, in particular Pax3d and Pax7B. Our results suggest that both Pax3 and Pax7 transcripts are required for commitment of cells to the myogenic lineage, with each transcript having a distinct role. More specifically, the Pax3c isoform may be required for terminal myogenic differentiation whereas the Pax3d isoform may be involved in undifferentiated cell maintenance and/or proliferation.

Mechanism of action of somatic stem cell treatments: towards the concept of therapeutic plasticity

The initial perception that transplantation of stem cells could serve only to replace damaged cells has continued to be modifi ed as evidence for a protective multifaceted function of transplanted cells has emerged. Neural stem/precursor cells (NPC) were the fi rst to be shown to use this strategy to protect the central nervous system (CNS) from infl ammatory insults (1 – 5). Subsequently similar functions by other types of somatic stem cells (e.g. mesenchymal stromal cells; MSC) have been described (6). Currently the ability of transplanted stem cells to protect tissues from diverse injury using multifaceted ‘ bystander strategies ’ has been substantiated in several experiments. Somatic stem cells promote CNS repair following intralesional as well as systemic injection by producing in situ a milieu of protective molecules whose release is regulated, both temporally and spatially, by environmental demands. The milieu contains immunomodulatory substances, trophic growth factors, stem cell regulators, angiogenic factors, scavengers, etc.: all molecules constitutively expressed by somatic stem cells for maintaining tissue homeostasis during development and in adult life. The concept of ‘ therapeutic platicity ’ that is now emerging (7) also reconciles data showing that stem cells other than NPC, although endowed with negligible transdifferentiation capability, may have a benefi cial role in CNS repair. In four articles of this issue of Cytotherapy the concept of therapeutic plasticity of somatic stem cells is presented further. Although these reports do not completely clarify the mechanisms underlying this therapeutic plasticity, they demonstrate that transplantation of various somatic stem cells via different routes ameliorates diverse neurologic diseases without an overt terminal differentiation and functional integration of the transplanted cells. Alexanian et al. (8) observe that delayed intralesional transplantation of human glial-restricted NPC in rats affected by thoracic spinal cord injury ameliorates sensorimotor defi cits. He et al. (9) report that bone marrow (BM)-derived MSC and endothelial progenitor cells transplanted intravenously (i.v.) protect rats from focal ischemia via the secretion of different combinations of growth factors. Interestingly, an additive benefi cial effect was observed when the two different cell sources were co-transplanted. Li et al. (10) focus on a mouse model of muscular dystrophy where mean survival as well as loss of dystrophin and utrophin expression is delayed by i.v. transplantation of rat-derived MSC. Attar et al. (11) describe preliminary results of intralesional transplantation of BM-derived hematopoietic stem cells along with BM-derived mononuclear cells in four patients with spinal cord injury. Although the possibility of using bystander strategies as protection from tissue injury appears to be a very promising therapeutic use of somatic stem cells, we still need to resolve unsolved key questions before clinical translation. One crucial question is whether to transplant the cells heterotopically or homotopically. Transplantation is not without risks: co-transplantation of NPC and pancreatic islet cells under the kidney capsule of diabetic mice induced the generation, at the site of cell transplantation, of insulin-induced neuroblastoma-like neoplasms (12). Intracardiac transplantation of stem cell factor-overexpressing MSC into mice with myocardial infarction promoted the formation of a chest wall-invading soft tissue sarcoma (13). The unregulated transplantation Cytotherapy, 2011; 13: 6–7

The Mesenchymal Stem Cell Antigen MSCA-1 is Identical to Tissue Non-specific Alkaline Phosphatase

We have recently identified 2 distinct CD271(bright)MSCA-1(dim)CD56(+) and CD271(bright)MSCA-1(bright)CD56(-) MSC subsets in primary femur-derived bone marrow (BM), which differ in their expression pattern and morphology as well as in their clonogenic and differentiation capacity. Here, we show that MSCA-1 is identical to tissue non-specific alkaline phosphatase (TNAP), an ectoenzyme known to be expressed at high levels in liver, bone, and kidney as well as in embryonic stem (ES) cells. SDS-PAGE of WERI-RB-1 cell lysate and supernatant from phosphatidylinositol-specific phospholipase C (PI-PLC)-treated WERI-RB-1 cells resulted in the appearance of a prominent 68-kDa band. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOF MS) sequence analysis revealed TNAP-specific peptides. Screening of the MSCA-1-specific antibody W8B2 on HEK-293 cells transfected with the full-length coding sequence of TNAP showed specific reactivity with transfected but not with parent cell line. In addition, TNAP-specific mRNA expression was selectively detected in the transfectant line. In agreement with these findings, enzymatic activity of TNAP was exclusively detected in sorted MSCA-1(+) BM cells but not in the MSCA-1(-) negative fraction. Surface marker analysis revealed coexpression of the embryonic marker SSEA-3 but not SSEA-4, TRA-1-60, and TRA-1-81. In endometrium, TNAP is expressed at intermediate levels on CD146(+) cells and at high levels in the luminal space of glandular epithelia. Our results demonstrate that TNAP is a selective marker for the prospective isolation of BM-derived MSC and MSC-like cells in endometrium.