Differentiation Potential Of Mesenchymal Stem Cells Research Articles

CD49f Acts as an Inflammation Sensor to Regulate Differentiation, Adhesion, and Migration of Human Mesenchymal Stem Cells.

The advent of mesenchymal stem cell (MSC)-based therapies has been an exciting innovation for the treatment of degenerative and inflammatory diseases. However, the surface markers that accurately reflect the self-renewal and differentiation potential of MSCs and their sensitivity to environmental cues remain poorly defined. Here, we studied the role of CD49f in bone marrow MSCs (BMSCs) and the mechanism by which it regulates the behavior of BMSCs under inflammatory conditions. We found that CD49f is preferentially expressed in fetal cells rather than adult cells, CD49f-positive BMSCs possess higher CFU-F formation ability and differentiation potential than CD49f negative cells, and the CD49f expression of BMSCs gradually decreases during in vitro passaging. CD49f knockdown dramatically decreased the differentiation of BMSCs and isoform A was demonstrated to be the main functional form that enhanced the differentiation ability of BMSCs. The influences of inflammatory cytokines on BMSCs revealed that TNF-α downregulated CD49f in BMSCs with impaired differentiation, decreased adhesion to laminins, and increased migration. Moreover, tissue transglutaminase was found to work together with CD49f to regulate the behavior of BMSCs. Finally, we showed that mTOR signaling rather than NF-κB activation mediated CD49f downregulation induced by TNF-α and maintained CD49f homeostasis in BMSCs. Our findings suggest that CD49f is a stemness marker of BMSCs and is tightly correlated with the behavioral changes of BMSCs under inflammatory conditions. These data demonstrate a novel role for CD49f in sensing inflammation through mTOR pathway to further modulate the behavior of MSCs to fulfill the requirements of the body.

Human Umbilical Cord-Derived Mesenchymal Stem Cells Improve Learning and Memory Function in Hypoxic-Ischemic Brain-Damaged Rats via an IL-8-Mediated Secretion Mechanism Rather than Differentiation Pattern Induction

Background: MSCs are a promising therapeutic resource. Paracrine effects and the induction of differentiation patterns are thought to represent the two primary mechanisms underlying the therapeutic effects of mesenchymal stem cell (MSC) transplantation in vivo. However, it is unclear which mechanism is involved in the therapeutic effects of human umbilical cord-derived MSC (hUC-MSC) transplantation. Methods and Results: Based on flow cytometry analysis, hUC-MSCs exhibited the morphological characteristics and surface markers of MSCs. Following directed neural induction, these cells displayed a neuron-like morphology and expressed high levels of neural markers. All types of hUC-MSCs, including differentiated and redifferentiated cells, promoted learning and memory function recovery in hypoxic-ischemic brain damaged (HIBD) rats. The hUC-MSCs secreted IL-8, which enhanced angiogenesis in the hippocampus via the JNK pathway. However, the differentiated and redifferentiated cells did not exert significantly greater therapeutic effects than the undifferentiated hUC-MSCs. Conclusion: hUC-MSCs display the biological properties and neural differentiation potential of MSCs and provide therapeutic advantages by secreting IL-8, which participates in angiogenesis in the rat HIBD model. These data suggest that hUC-MSC transplantation improves the recovery of neuronal function via an IL-8-mediated secretion mechanism, whereas differentiation pattern induction was limited.

Abstract 5420: Nano-engineered mesenchymal stem cells as targeted therapeutic carriers

Abstract Poor availability of chemotherapeutics in deep-seated and metastatic cancers results in the development of drug resistance and failed therapeutic outcomes. Approaches that can specifically enhance drug delivery to the tumor tissue can potentially improve therapeutic efficacy while minimizing toxic side effects. In our current studies, we used nano-engineered mesenchymal stem cells (MSCs) as tumor-targeted therapeutic carriers. MSCs possess exquisite tumor homing capabilities. In addition, MSCs overexpress efflux transporters such as P-glycoprotein and are highly drug resistant. The inherent tumor-tropic and drug-resistant properties make MSCs ideal carriers for toxic payload. Since a majority of MSCs first accumulate in the lungs following systemic administration, this technology is particularly suitable for the delivery of anticancer therapeutics to lung tumors. The overall objective of this study was to investigate the efficacy of nano-engineered MSCs in non-small cell lung cancer. We hypothesized that incorporation of drug-loaded, polymeric nanoparticles in MSCs will allow for tumor-targeted and sustained drug delivery. Nanoparticles containing paclitaxel and imaging agent were formulated using poly (DL-lactide-co-glycolide) polymer by solvent evaporation technique and characterized for size, charge, drug loading and in vitro drug release. Nano-engineered MSCs were designed by loading MSCs with nanoparticles containing paclitaxel and an infrared (in vivo) or green fluorescent (in vitro) imaging agent and characterized for nanoparticle uptake using flow cytometry. Nano-engineered MSCs were characterized for in vitro cytotoxicity, differentiation potential and in vivo migratory potential. Human MSCs incubated with nanoparticles showed a steady increase in nanoparticle uptake, with saturation at 4 hours. Nanoparticles encapsulation did not affect the viability or the differentiation potential of MSCs. Nano-engineered MSCs resulted in dose-dependent cytotoxicity in A549 lung adenocarcinoma cells. An orthotopic lung tumor model was used to monitor the in vivo distribution of nano-engineered MSCs. Intravenous injection of A549 cells stably transfected with luciferase in mice resulted in formation of lung tumors that were monitored by bioluminescence imaging. Intravenous injection of nanoparticles alone resulted in non-specific biodistribution, with high accumulation in liver and spleen. Nano-engineered MSCs, in contrast, demonstrated selective accumulation and retention in lung tumors. These results indicate that nano-engineered MSCs can be loaded with high concentration of anticancer agents without affecting their migratory or drug resistance properties. Future studies will examine the anticancer efficacy of nano-engineered MSCs in an orthotopic mouse model of lung cancer. Citation Format: Swayam Prabha, Tanmoy Sadhukha. Nano-engineered mesenchymal stem cells as targeted therapeutic carriers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5420. doi:10.1158/1538-7445.AM2014-5420

Downregulated CXCL12 expression in mesenchymal stem cells associated with severe aplastic anemia in children.

The mechanisms of idiopathic severe aplastic anemia (SAA) in children are not completely understood. Insufficiency of the bone marrow microenvironment, in which mesenchymal stem cells (MSCs) are an important element, can be a potential factor associated with hematopoietic impairment. In the current study, we studied whether aberrant gene expression could be found in MSCs from children with SAA. Using microarray analysis, two different patterns of global gene expression were detected in the SAA MSCs. Fourteen genes (POLE2, HGF, KIF20A, TK1, IL18R1, KITLG, FGF18, RRM2, TTK, CXCL12, DLG7, TOP2A, NUF2, and TYMS), which are related to DNA synthesis, cytokines, or growth factors, were significantly downregulated. Further, knockdown of gene expression was performed using the small hairpin RNA (shRNA)-containing lentivirus method. We found that knockdown of CXCL12, HGF, IL-18R1, FGF18, or RRM2 expression compelled MSCs from the controls to behave like those from the SAA children, with decreased survival and differentiation potential. Among them, inhibition of CXCL12 gene expression had the most profound effects on the behavior of MSCs. Further experiments regarding re-introduction of the CXCL12 gene could largely recover the survival and differentiation potential in MSCs with inhibition of CXCL12 expression. Our findings suggest that MSCs from children with SAA exhibit aberrant gene expression profiles and downregulation of CXCL12 gene may be associated with alterations in the bone marrow microenvironment.

Senescence suppressors: their practical importance in replicative lifespan extension in stem cells.

Recent animal and clinical studies report promising results for the therapeutic utilization of stem cells in regenerative medicine. Mesenchymal stem cells (MSCs), with their pluripotent nature, have advantages over embryonic stem cells in terms of their availability and feasibility. However, their proliferative activity is destined to slow by replicative senescence, and the limited proliferative potential of MSCs not only hinders the preparation of sufficient cells for in vivo application, but also draws a limitation on their potential for differentiation. This calls for the development of safe and efficient means to increase the proliferative as well as differentiation potential of MSCs. Recent advances have led to a better understanding of the underlying mechanisms and significance of cellular senescence, facilitating ways to manipulate the replicative lifespan of a variety of primary cells, including MSCs. This paper introduces a class of proteins that function as senescence suppressors. Like tumor suppressors, these proteins are lost in senescence, while their forced expression delays the onset of senescence. Moreover, treatments that increase the expression or the activity of senescence suppressors, therefore, cause expansion of the replicative and differentiation potential of MSCs. The nature of the activities and putative underlying mechanisms of the senescence suppressors will be discussed to facilitate their evaluation.

Carfilzomib promotes the osteogenic differentiation potential of mesenchymal stem cells derived from myeloma patients by inhibiting notch1 activity in vitro

Notch1 signaling plays a key role in the differentiation of mesenchymal stem cells (MSCs). Carfilzomib (CFZ), a second-generation proteasome inhibitor, has potent cytotoxicity against myeloma cells. In this study, we investigated the effects of CFZ on the osteogenic differentiation potential of MSCs derived from myeloma patients (MM-MSCs) in vitro. MM-MSCs showed decreased osteogenic differentiation ability, together with an impairment of notch1 deactivation. The notch1 inhibitor DAPT and the downregulation of notch1 by shRNA promoted osteogenesis in MM-MSCs. Additionally, CFZ treatment resulted in notch1 inhibition and enhanced osteogenesis in MM-MSCs. These findings suggest that CFZ stimulates osteogenesis via notch1 inhibition.

Electro-acupuncture promotes the survival and differentiation of transplanted bone marrow mesenchymal stem cells pre-induced with neurotrophin-3 and retinoic acid in gelatin sponge scaffold after rat spinal cord transection.

In the past decades, mesenchymal stem cells (MSCs) as a promising cell candidate have received the most attention in the treatment of spinal cord injury (SCI). However, due to the low survival rate and low neural differentiation rate, the grafted MSCs do not perform well as one would have expected. In the present study, we tested a combinational therapy to improve on this situation. MSCs were loaded into three-dimensional gelatin sponge (GS) scaffold. After 7days of induction with neurotrophin-3 (NT-3) and retinoic acid (RA) in vitro, we observed a significant increase in TrkC mRNA transcription by Real-time PCR and this was confirmed by in situ hybridization. The expression of TrkC was also confirmed by Western blot and immunohistochemistry. Differentiation potential of MSCs in vitro into neuron-like cells or oligodendrocyte-like cells was further demonstrated by using immunofluorescence staining. The pre-induced MSCs seeding in GS scaffolds were then grafted into the transected rat spinal cord. One day after grafting, Governor Vessel electro-acupuncture (GV-EA) treatment was applied to rats in the NR-MSCs + EA group. At 30days after GV-EA treatment, it found that the grafted MSCs have better survival rate and neuron-like cell differentiation compared with those without GV-EA treatment. The sustained TrkC expression in the grafted MSCs as well as increased NT-3 content in the injury/graft site by GV-EA suggests that NT-3/TrkC signaling pathway may be involved in the promoting effect. This study demonstrates that GV-EA and pre-induction with NT-3 and RA together may promote the survival and differentiation of grafted MSCs in GS scaffold in rat SCI.

Substance P modulates properties of bone marrow-derived mesenchymal stem cells

Bone marrow derived mesenchymal stem cells (MSCs) have self-renewal characteristics and are able to differentiate into various cell types. These cells can mobilize to peripheral tissues in order to participate in important biological processes such as wound healing. They are also able to regulate hematopoietic stem cells (HSCs) trafficking between the bone marrow and the periphery through the regulation of intercellular interactions and the expression of cytokines, such as SDF-1. Previous studies demonstrated that substance P (SP) induces the proliferation of MSCs in vitro and mediates the migration of MSCs to injury sites to promote healing. We found that SP increases CFU-F of the bone marrow total cells and up-regulates the osteogenic differentiation potential of MSCs without affecting their adipogenic differentiation potential. We also found that SP increases the mRNA levels of SDF-1 and N-cadherin in total bone marrow. Therefore, it is very likely that SP modulates the properties of MSCs linked to their therapeutic potential and their role in trafficking of stem cells.

Characterization of the alterations in the differentiation potential of porcine mesenchymal stem cell caused by neonatal dietary calcium nutrition (819.2)

Neonatal Ca nutrition affects bone development and mesenchymal stem cell (MSC) activity; however, its effect on MSC differentiation potential has not been well characterized. We assessed the differentiation potential of both the heterogenous MSC population as well as the individual clones isolated from neonatal pigs (n=5) fed either a Ca deficient, adequate, or excessive diet. The most and least osteogenic (O+/O‐) and the most and least adipogenic (A+/A‐) clones were selected from each pig by functional staining. The MSC from Ca deficient pigs had reduced osteogenic potential and enhanced adipogenic potential based on functional staining and the gene expression of osteocalcin (OC), PPARG, and AP2 compared with MSC from Ca adequate pigs. Interestingly, MSC from pigs receiving excess Ca also had increased adipogenic potential. Evaluation of clonal isolates indicated that the O+ clones from Ca deficient pigs had reduced OC expression (P < 0.05), and O‐ clones from the Ca excess pigs had increased gene expression of both PPARG and AP2. Among the A+ clones of both Ca deficient and excess pigs, Runx2 expression was decreased (P < 0.05). These data suggest that the alterations in the differentiation potential of MSC based on neonatal Ca status of the pig may be caused by changes in both the lineage priming and the lineage allocation of MSC.

In vitro and in vivo neurogenic potential of mesenchymal stem cells isolated from different sources

Regenerative medicine is an evolving interdisciplinary topic of research involving numerous technological methods that utilize stem cells to repair damaged tissues. Particularly, mesenchymal stem cells (MSCs) are a great tool in regenerative medicine because of their lack of tumorogenicity, immunogenicity and ability to perform immunomodulatory as well as anti-inflammatory functions. Numerous studies have investigated the role of MSCs in tissue repair and modulation of allogeneic immune responses. MSCs derived from different sources hold unique regenerative potential as they are self-renewing and can differentiate into chondrocytes, osteoblasts, adipocytes, cardiomyocytes, hepatocytes, endothelial and neuronal cells, among which neuronal-like cells have gained special interest. MSCs also have the ability to secrete multiple bioactive molecules capable of stimulating recovery of injured cells and inhibiting inflammation. In this review we focus on neural differentiation potential of MSCs isolated from different sources and how certain growth factors/small molecules can be used to derive neuronal phenotypes from MSCs. We also discuss the efficacy of MSCs when transplanted in vivo and how they can generate certain neurons and lead to relief or recovery of the diseased condition. Furthermore, we have tried to evaluate the appropriatemerits of different sources ofMSCs with respect to their propensity towards neurological differentiation as well as their effectiveness in preclinical studies.

Poly(Epsilon-Lysine) Dendrons Tethered with Phosphoserine Increase Mesenchymal Stem Cell Differentiation Potential of Calcium Phosphate Gels

Calcium phosphates (CaP) are considered as biomaterials of choice for the treatment of critical-sized bone defects. Novel injectable CaP materials integrating poly(epsilon-lysine) generation 3 dendrons tethered with phosphoserine were obtained by sol-gel synthesis. This type of dendron was integrated to mimic the biochemical structure of noncollagenous proteins present in the forming osteoids during bone repair. Sol-gel synthesis was coupled with a dialysis process able to equilibrate the materials at a physiological pH value. Fourier transform infrared spectroscopy (FTIR) showed the successful retention of the dendrons after gel dialysis, whereas X-ray diffraction analysis demonstrated both the pH-tuned formation of a hydroxyapatite crystalline phase within the gel and the complete removal of ammonium nitrate deriving from the sol-gel reaction solvent. Scanning electron microscopy images confirmed the presence of crystalline domains in gels synthesized at pH 9.0. Injectability tests showed that the optimized formulations fulfilled the rheological properties required to minimally invasive surgical procedures. Cytotoxicity tests on osteoblast-like MG-63 cells as well as morphology and viability studies showed that the dendrons induced a significantly higher level of cell proliferation at early incubation time. Differentiation of the cell was also clearly enhanced at longer incubation time as demonstrated by both alkaline phosphatase activity and expression of typical markers. Altogether, the data from this work indicate the clinical potential of the osteoid-mimicking CaP cements in minimally invasive bone surgery.

Human Bone Marrow-Derived Mesenchymal Stem Cells Display Enhanced Clonogenicity but Impaired Differentiation With Hypoxic Preconditioning

Stem cells are promising candidate cells for regenerative applications because they possess high proliferative capacity and the potential to differentiate into other cell types. Mesenchymal stem cells (MSCs) are easily sourced but do not retain their proliferative and multilineage differentiative capabilities after prolonged ex vivo propagation. We investigated the use of hypoxia as a preconditioning agent and in differentiating cultures to enhance MSC function. Culture in 5% ambient O(2) consistently enhanced clonogenic potential of primary MSCs from all donors tested. We determined that enhanced clonogenicity was attributable to increased proliferation, increased vascular endothelial growth factor secretion, and increased matrix turnover. Hypoxia did not impact the incidence of cell death. Application of hypoxia to osteogenic cultures resulted in enhanced total mineral deposition, although this effect was detected only in MSCs preconditioned in normoxic conditions. Osteogenesis-associated genes were upregulated in hypoxia, and alkaline phosphatase activity was enhanced. Adipogenic differentiation was inhibited by exposure to hypoxia during differentiation. Chondrogenesis in three-dimensional pellet cultures was inhibited by preconditioning with hypoxia. However, in cultures expanded under normoxia, hypoxia applied during subsequent pellet culture enhanced chondrogenesis. Whereas hypoxic preconditioning appears to be an excellent way to expand a highly clonogenic progenitor pool, our findings suggest that it may blunt the differentiation potential of MSCs, compromising their utility for regenerative tissue engineering. Exposure to hypoxia during differentiation (post-normoxic expansion), however, appears to result in a greater quantity of functional osteoblasts and chondrocytes and ultimately a larger quantity of high-quality differentiated tissue.

Osteogenic lineage commitment of mesenchymal stem cells from patients with ossification of the posterior longitudinal ligament

Ectopic bone formation is thought to be responsible for ossification of the posterior longitudinal ligament of the spine (OPLL). Mesenchymal stem cells (MSCs) were isolated from spinal ligaments and shown to play a key role in the process of ectopic ossification. The purpose of this study was to explore the capacity of these MSCs to undergo lineage commitment and to assess the gene expression changes between these committed and uncommitted MSCs between OPLL and non-OPLL patients. Spinal ligament-derived cells were obtained from OPLL patients or patients with cervical spondylotic myelopathy (non-ossified) for comparison (n=8 in each group). MSCs from the two patient cohorts were evaluated for changes in colony forming ability; osteogenic, adipogenic and chondrogenic differentiation potential; and changes in gene expression following induction with lineage-specific conditions. We show that the osteogenic differentiation potential was significantly higher in MSCs from OPLL patients than in those from non-OPLL patients. In addition, alkaline phosphatase activity and several osteogenic-related genes expressions (bone morphogenetic protein 2, runt-related transcription factor 2 and alkaline phosphatase) were significantly higher in the OPLL group than in the non-OPLL group. However, single cell cloning efficiency, adipogenic and chondrogenic differentiation, and the expression of adipogenic and chondrogenic-related genes were equivalent between MSCs harvested from OPLL and non-OPLL patient samples. These findings suggest an increase in the osteogenic differentiation potential of MSCs from OPLL patients and that this propensity toward the osteogenic lineage may be a causal factor in the ossification in these ligaments.

The Supportive Role Of Mesenchymal Stem Cells On Normal Hematopoietic Stem Cells Is Impaired In Leukemic Bone Marrow

Mesenchymal stem cells (MSCs) have been shown to be able to form a niche that maintains hematopoietic stem cells (HSCs). However, how the phonotypical characteristics and biological functions of MSCs are affected and especially whether their supporting role on normal HSC is impaired in leukemic microenvironment, are not well defined. Here, we have examined the changes of biological characteristics of mouse MSCs and in particular their supporting function on normal HSCs in Notch1-induced acute T cell lymphocytic leukemia (T-ALL) mice (Hu et al, Blood 2009; 114:3783-3792). MSCs were sorted based on the phenotypic markers, PDGFRα+Sca-1+CD45-TER119- (PαS) (Houlihan et al, Nat Protoc 2012) from control or T-ALL mice and flow cytometric analysis was conducted at different time points during leukemia development. Our results showed there was no significant difference between T-ALL and control mice regarding the absolute number and percentage of PαS MSCs in total bone marrow mono-nuclear cells (BMMNCs), cell cycling status and the percentage of apoptosis of freshly sorted PαS MSCs. Moreover, PαS MSCs from both normal and T-ALL leukemic marrow had similar morphology (spindle and polymorphic shaped), and uniformly expressed known cell surface markers for cultured mouse MSCs (CD29, CD44, Sca-1). However, the number of T-ALL PαS MSCs colony forming unit-fibroblastic (CFU-F) formation was 3-fold lower than control CFU-F (p<0.001). Levels of mRNA expression of genes associated with adipogenic (adipsin, mLP, PPARγ), osteogenic (Bmp4, Sp7, Bglap) and chondrogenic (collagen II, collagen X, aggrecan) differentiation in PαS MSCs were significantly decreased in T-ALL mice compared with control mice (p<0.05). These results suggest that the proliferation potential and the differentiation potential of MSC were decreased in the leukemic environment. To investigate whether the decrease in the proliferation of T-ALL PαS MSCs was associated with a change in cellular senescence, β-galactosidase activity and quantitative RT-PCR analysis of genes associated with senescence were performed. Our finding showed a significant increase in the number of β-galactosidase–positive cells (control/T-ALL; 3.11±0.20%/3.99±0.08%, p<0.05) and in the mRNA expression level of senescence-related gene p16 in PαS MSCs T-ALL mice compared with control mice (control/T-ALL; 1.00±0.03/1.57±0.17, p<0.05). These results imply that p16 plays an important role in PαS MSCs senescence in T-ALL microenvironment. To determine whether the supporting function of T-ALL PαS MSCs on normal HSCs proliferation is impaired, the cobble-stone area forming cell (CAFC) assay, an in vitro surrogate for HSCs, was applied. Normal control or T-ALL PαS MSCs were co-cultured with the same pool of normal HSCs. After 5 weeks, co-cultures containing normal PαS MSC formed multiple large CAFCs. On the contrast, T-ALL PαS MSCs supported 7-fold less hypocellular CAFCs (p<0.05). This data indicate that T-ALL PαS MSCs have a reduced ability to support normal HSCs proliferation in vitro. To further validate the change of PαS MSCs' supporting function on normal HSCs proliferation in leukemic microenvironment observed in vitro, in vivo co-transplantation study were performed. Our preliminary results further indicated that the supporting function of MSCs on normal hematopoiesis in the leukemic microenvironment was compromised. In conclusion, dysfunction of MSC, an important component of HSC microenvironment, may play a crucial role in the suppression of HSC during leukemia development. Improving the function of MSC may serve as a new strategy to enhance normal hematopoiesis in leukemic marrow. Disclosures:No relevant conflicts of interest to declare.

Periostin Contributes to the Acquisition of Multipotent Stem Cell-Like Properties in Human Mammary Epithelial Cells and Breast Cancer Cells

Periostin (POSTN), a recently characterised matricellular protein, is frequently dysregulated in various malignant cancers and promotes tumor metastatic growth. POSTN plays a critical role in the crosstalk between murine breast cancer stem cells (CSCs) and their niche to permit metastatic colonization. However, whether pro-metastatic capability of POSTN is associated with multipotent potentials of mesenchymal stem cells (MSCs) has not been documented. Here we demonstrate that POSTN promotes a stem cell-like trait and a mesenchymal phenotype in human mammary epithelial cells and breast cancer cells. Interestingly, ectopic overexpression of POSTN or recombinant POSTN treatment can induce human mammary epithelial cells and breast cancer cells differentiation into multiple cell lineages that recapitulate part of the multilineage differentiation potentials of MSCs. Moreover, POSTN is highly expressed in bone marrow-derived MSCs and their derived adipocytes, chondrocytes, and osteoblasts in vitro. Furthermore, POSTN promotes the growth of xenograft tumors in vivo. POSTN-overexpressing human mammary epithelial cells enhance breast tumor growth and metastasis. These data thus provide evidence of a new role for POSTN in mammary epithelial neoplasia and metastasis, suggesting that epithelial cancer cells might acquire CSC-like traits and a mesenchymal phenotype, as well as the multipotent potentials of MSCs to promote tumorigenesis and metastasis. Therefore, targeting POSTN and other extracellular matrix components of tumor microenvironment may help to develop new therapeutical strategies to inhibit tumor metastasis.

Fibrin Glue Improves the Therapeutic Effect of MSCs by Sustaining Survival and Paracrine Function

Fibrin glue has been widely investigated as a cell delivery vehicle for improving the therapeutic effects of mesenchymal stem cells (MSCs). Implanted MSCs produce their therapeutic effects by secreting paracrine factors and by replacing damaged tissues after differentiation. While the influence of fibrin glue on the differentiation potential of MSCs has been well documented, its effect on paracrine function of MSCs is largely unknown. Herein we investigated the influence of fibrin glue on the paracrine effects of MSCs. MSCs were isolated from human adipose tissue. The effects of fibrin glue on survival, migration, secretion of growth factors, and immune suppression of MSCs were investigated in vitro. MSCs in fibrin glue survived and secreted growth factors such as the vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) over 14 days. VEGF and immune modulators, including the transforming growth factor (TGF)-β1 and prostaglandin E2, secreted from MSCs in fibrin glue significantly increased under inflammatory conditions. Thus, MSCs in fibrin glue effectively suppressed immune reactions. In addition, fibrin glue protected the MSCs from oxidative stress and prevented human dermal fibroblast death induced by exposure to extreme stress. In contrast, MSCs within fibrin glue hardly migrated. These results suggest that fibrin glue may sustain survival of implanted MSCs and their paracrine function. Our results provide a mechanistic data to allow further development of MSCs with fibrin glue as a clinical treatment.

Microwell Chips for Selection of Bio‐macromolecules that Increase the Differentiation Capacities of Mesenchymal Stem Cells

Microwell chips (25 mm × 25 mm) are fabricated to select proper substrates for growing three-dimensional (3D) spheroids from mesenchymal stem cells (MSCs). Different bio-macromolecules and their combinations are immobilized on the chip by air plasma treatment and by polyelectrolyte interaction. Only a small number of MSCs (≈10(5) ) are needed for each chip. The expression level of N-cadherin, a cell-adhesion molecule, is used as an indicator for cell-cell interactions. MSC spheroids expressing the highest N-cadherin level also show the greatest osteogenic potential. The microwell chip may be used as an efficient platform to screen bio-macromolecules that enhance the differentiation potential of MSCs.

Neonatal dietary calcium alters mesenchymal stem cell differentiation potential in pigs

Neonatal Ca nutrition affects bone development and mesenchymal stem cell (MSC) activity; however, its effect on MSC differentiation potential has not been fully characterized. MSC were isolated from neonatal pigs fed either a Ca deficient, adequate, or excessive diet. After in vitro adipogenic induction, PPARG and AP2 expression was up‐regulated in MSC from Ca deficient (P&lt;0.1) and Ca excess (P&lt;0.05) groups. Under osteogenic induction, osteocalcin (OC) gene expression was depressed in MSC from Ca deficient pigs. To further evaluate the impact of Ca status on MSC activity, 20 individual colonies were obtained from each pig. From these colonies, we selected the most and least osteogenic (O+/O−) and the most and least adipogenic (A+/A−) clones based on functional staining. During osteogenesis, increased (P &lt; 0.1) gene expression of Runx2 and OC was seen among O+ colonies. Increased expression of OC was seen among Ca adequate (P&lt;0.01) and excess (P=0.07) colonies compared with the Ca deficient colonies. During adipogenesis, A+ colonies had greater expression (P &lt; 0.1) of AP2 and LPL. No interaction between osteogenic or adipogenic capability and Ca status was seen. This suggests that the differences in adipocytic and osteogenic differentiation of MSC based on the Ca status of the pig is due to alterations in the number of highly osteogenic and adipogenic MSC in the heterogeneous population.Grant Funding Source: NC Agricultural Research Service

New Frontiers in Regenerative Medicine in Cardiology: The Potential of Wharton’s Jelly Mesenchymal Stem Cells

Cardiomyopathies are still the first cause of death in the world. The identification of resident stem cells, comprising those derived from sub-endocardial stroma, suggests the possible self regeneration of the heart under autocrine/paracrine modulation in the cardiac microenvironment. Nevertheless, because of the limited in vivo regeneration potential of damaged cardiac tissue, the use of drugs and ultimately cardiac transplantation remain the common treatments of heart diseases and defects. The differentiative potential of embryonic and mesenchymal stem cells (MSCs) derived from different tissues (such as bone marrow and adipose tissue) was extensively explored in cell therapy for regenerative medicine. Many groups have been focused, in recent years, on isolation, characterization, and differentiation potential of MSCs derived from perinatal (or extraembryonic) tissues, mainly the placenta and the human umbilical cord. In this review, we summarized recent works about the stemness of Wharton's jelly stromal cells and their potential in cardiac regeneration with favourable use in cell therapy and regenerative medicine. The peculiar features of these cells, as the expression of cardiac-specific transcription factors and immunomodulatory molecules suggest that human umbilical cord may be considered as a reliable alternative source of MSC useful for advanced therapy in cardiac regenerative medicine.

Pigment epithelium‐derived factor delays cellular senescence of human mesenchymal stem cells in vitro by reducing oxidative stress

Mesenchymal stem cells (MSCs) are multipotent progenitor cells that represent a promising approach in the field of regenerative medicine; however, this potential diminishes with senescence. Pigment epithelium-derived factor (PEDF) gives some protection by reducing oxidative stress, which is known to accelerate cellular senescence. Thus we hypothesized that PEDF could delay senescence during MSC expansion by reducing oxidative stress. Proliferation and differentiation potentials, oxidative stress, senescence and p53/p16 expressions have been examined. In MSCs cultured under normoxic conditions treated with PEDF, proliferative lifespan in vitro was significantly increased compared with control group not given PEDF, with ∼10 additional population doublings (PD) occurring before terminal growth arrest. Most of the MSCs cultured under normoxic conditions ceased to proliferate after 20-28 PD, while few senescent cells were found in the hypoxic, PEDF-hypoxic and PEDF-normoxic cultures; this was associated with downregulation of p53 and p16 expression and decreased oxidative stress. PEDF also preserved differentiation potentials of MSCs compared with the control group. Thus PEDF suppression of oxidative stress delays cellular senescence and allows greater expansion of MSCs.