Cord Blood-derived Mesenchymal Stem Cells Research Articles

Mesenchymal stem cells for the prevention of bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) is a chronic lung disease in preterm infants who have been treated with supplemental oxygen and mechanical ventilation. Despite major advances in perinatal and neonatal medicine, limited progress has been made in reducing BPD rates. The use of mesenchymal stem cells (MSC) is a promising and innovative therapy for several diseases because they are easy to extract and they have low immunogenicity, anti-inflammatory properties, and regenerative ability. According to several pre-clinical studies that have used BPD animal models, one mechanism of action for MSC in BPD is mainly due to the paracrine effects of MSC-derived humoral factors, such as interleukin (IL)-6, IL-8, vascular endothelial growth factor, collagen, and elastin, rather than the multilineage and regenerative capacities of MSC. Cell-free preparations derived from MSC, including conditioned media and exosomes, remain a pre-clinical technology despite their great clinical potential. A first-in-human clinical trial of MSC treatment for BPD was performed as a phase I dose-escalation trial using umbilical cord blood-derived MSC. That trial demonstrated the short- and long-term safety and feasibility of MSC, given that significantly reduced inflammatory marker expression was observed in tracheal aspirates. As of recently, several clinical trials of MSC use for BPD are ongoing or are planned in some countries to investigate the efficacy of MSC in the prevention or treatment of BPD in premature infants. Many clinicians are currently awaiting the results from these trials so that MSC can be used clinically for human BPD.

Decorin Secreted by Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Induces Macrophage Polarization via CD44 to Repair Hyperoxic Lung Injury.

Bronchopulmonary dysplasia (BPD), caused by hyperoxia in newborns and infants, results in lung damage and abnormal pulmonary function. However, the current treatments for BPD are steroidal and pharmacological therapies, which cause neurodevelopmental impairment. Treatment with umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) is an efficient alternative approach. To prevent pulmonary inflammation in BPD, this study investigated the hypothesis that a key regulator was secreted by MSCs to polarize inflammatory macrophages into anti-inflammatory macrophages at inflammation sites. Lipopolysaccharide-induced macrophages co-cultured with MSCs secreted low levels of the inflammatory cytokines, IL-8 and IL-6, but high levels of the anti-inflammatory cytokine, IL-10. Silencing decorin in MSCs suppressed the expression of CD44, which mediates anti-inflammatory activity in macrophages. The effects of MSCs were examined in a rat model of hyperoxic lung damage. Macrophage polarization differed depending on the levels of decorin secreted by MSCs. Moreover, intratracheal injection of decorin-silenced MSCs or MSCs secreting low levels of decorin confirmed impaired alveolarization of damaged lung tissues by down-regulation of decorin. In tissues, a decrease in the anti-inflammatory macrophage marker, CD163, was observed via CD44. Thus, we identified decorin as a key paracrine factor, inducing macrophage polarization via CD44, a master immunoregulator in mesenchymal stem cells.

Enhanced chondrogenic differentiation of human mesenchymal stem cells in silk fibroin/chitosan/glycosaminoglycan scaffolds under dynamic culture condition

Cartilage tissue damage and diseases are the most common clinical situation that occurs because of aging and injury, thereby causing pain and loss of mobility. The inability of cartilage tissue to self-repair is instrumental in developing tissue engineered substitutes. To this effect, the present study aims to engineer cartilage construct by culturing umbilical cord blood-derived human mesenchymal stem cells (hMSCs) on novel 3D porous scaffolds developed from natural biopolymers, silk fibroin (SF) and chitosan (CS), with addition of cartilage matrix components, glucosamine (Gl) and chondroitin sulfate (Ch). The presence of Gl and Ch is expected to enhance cartilage regeneration. The developed SF/CS-Gl-Ch scaffolds possess desired pore size in the range 56.55–168.15 μm, 88–92% porosity, 44.7–46.8̊ contact angle, controlled swelling and biodegradability. Upon culturing under dynamic condition in a spinner flask bioreactor, the scaffold supported hMSCs attachment, proliferation, and further promoted chondrogenic differentiation. Cartilage-specific matrix and gene (Collagen II, Sox9 and aggrecan) expression analyses by histology, immunophenotype, immunofluorescence and quantitative PCR studies showed superiority of cell-scaffold construct generated in dynamic culture towards cartilage tissue generation as compared to cell aggregates formed by pellet culture. This study demonstrates the potentiality of SF/CS-Gl-Ch porous scaffold for the development of tissue construct for cartilage regeneration under dynamic culture condition.

The ERK signaling pathway is involved in cardiotrophin-1-induced neural differentiation of human umbilical cord blood mesenchymal stem cells in vitro.

Central nervous system diseases remain the most challenging pathologies, with limited or even no therapeutic possibilities and a poor prognosis. This study aimed to investigate the differentiation properties of human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) transfected with recombinant adenovirus expressing enhanced green fluorescence protein cardiotrophin-1 (Adv-EGFP-CT-1) and the possible mechanisms involved. Cells were isolated, and MSC immunophenotypes were confirmed. The resulting differentiated cells treated with Adv-EGFP-CT-1 and cultured in neural induction medium (NIM) expressed higher levels of Nestin, neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) markers than cells in other treatments. Expression of glycoprotein 130/leukemia inhibitory factor receptor β (gp130/LiFRβ), Raf-1, phosphorylated Raf-1 (p-Raf-1), extracellular signal-regulated kinase 1/2 (ERK1/2) and phospho-ERK1/2 (p-ERK1/2) increased gradually within 72h after transfection with Adv-EGFP-CT-1 and NIM culture. Additionally, inhibition of extracellular signal-regulated kinase kinase (MEK) abrogated expression of p-ERK1/2, Nestin, GFAP and NeuN. Thus, the ERK1/2 pathway may contribute to CT1-stimulated neural differentiation of hUCB-MSCs.

The impact of parathyroid hormone treated mesenchymal stem cells on ex-vivo expansion of cord blood hematopoietic stem cells

Ex-vivo expansion of cord blood hematopoietic stem cells (CB-HSCs) may play a pivotal role in the process of hematopoietic stem cell transplantation (HSCT). Here, we investigated the expansion of CB-HSCs and hematopoietic precursor cells (HPCs) on a mesenchymal feeder layer treated with Parathyroid hormone (PTH) and compared them with the untreated feeder layer. Cord blood mesenchymal and hematopoietic stem cells were used in four culture conditions in order to assess the ability of PTH to maintain HSCs self-renewability: 1) Co-culture with UC-MSCs treated feeder layer (CO + PTH), 2) Co-culture with untreated UC-MSCs feeder layer (CO-PTH), 3) Co-culture with cytokine cocktail and PTH without feeder layer (Cyto+PTH), 4) Co-culture with cytokine cocktail but without PTH and feeder layer (Cyto-PTH). After expansion, total nucleated cells (TNC) and CD34+ cells were quantified. In addition, colony-forming unit (CFU) assay and Long-Term Culture Initiating Cell (LTC-IC) were performed. Finally, expression of HES1 gene was assessed. In CO + PTH group HES1 gene, which is responsible for self-maintenance and self-renewal of CB-HSCs was over-expressed. Importantly, PTH treated cells could maintain both CFU and LTC-IC in CO + PTH group which reflects the impact of PTH treatment on UC-HSC expansion. Our study suggests that UC-MSCs feeder layer treated with PTH is able to expand CB-HSCs, which is critical for HSCT in adult patients.

Exosomal miR-1246 derived from human umbilical cord blood mesenchymal stem cells attenuates hepatic ischemia reperfusion injury by modulating T helper 17/regulatory T balance.

The purpose of this study was to explore the mechanism by which human umbilical cord blood mesenchymal stem cells (hUCB-MSCs)-derived exosomes exerted protective effect in hepatic ischemia/reperfusion injury (IRI). hUCB-MSCs-derived exosomes were administrated into hepatic IRI mice or cocultured with naïve CD4+ T cells exposed to hepatic hypoxia/reoxygenation microenvironment. Hepatic function was assessed by determining serum transaminases. Histological changes were observed using hematoxylin and eosin staining. The proportion of T helper 17 (Th17) and regulatory T (Treg) cells were analyzed by flow cytometry. The concentration of inflammatory cytokines was determined by enzyme-linked immunosorbent assay. The interaction between miR-1246 and interleukin 6 (IL-6) signal transducer (also known as gp130) was verified by luciferase activity assay. The miR-1246 expression, Th17/Treg-related genes, and gp130-signal transducer and activator of transcription 3 (STAT3) pathway were detected by quantitative real-time polymerase chain reaction and western blotting. hUCB-MSCs-derived exosomes ameliorated IRI-induced hepatic dysfunction and decreased Th17/Treg ratio in CD4+ T cells in vitro, whereas treatment of hUCB-MSCs with miR-1246 inhibitor showed opposite effects, which was mediated via the IL-6-gp130-STAT3 pathway. hUCB-MSCs-derived exosomes could alleviate hepatic IRI through modulating the balance between Tregs and Th17 cells via miR-1246-mediated IL-6-gp130-STAT3 axis.

Therapeutic effect of human umbilical cord blood mesenchymal stem cells combined with G-CSF on rats with acute liver failure

Human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) have been used to facilitate healing in animal models of liver injury, while granulocyte colony-stimulating factor (G-CSF) has been shown to stimulate stem cell mobilization and these cells may contribute to liver repair. hUCB-MSCs were characterized by flow cytometry, and transplanted into rats with d-galactosamine (D-GalN)/lipopolysaccharides (LPS)-induced acute liver failure (ALF) together with granulocyte colony-stimulating factor (G-CSF). Liver function, oxidative stress and pro-inflammatory cytokines expressions were examined using enzyme-linked immunosorbent assay (ELISA). Hematoxylin-eosin (HE) staining was used to observe the morphological changes. Apoptosis was investigated by terminal dUTP nick end labeling (TUNEL) staining. Bromodeoxyuridine (BrdU) cell proliferation assay was analyzed by immunofluorescence and immunohistochemistry. In the results, cultured hUCB-MSCs displayed proliferation and adipogenic and osteogenic differentiation potentials. hUCB-MSCs in combination with G-CSF significantly attenuated ALF-induced liver function injury. Furthermore, hUCB-MSCs and G-CSF treatment remarkably suppressed the secretions of pro-inflammatory cytokines and MDA activation induced by ALF. In addition, inflammation, lesions and cell apoptosis in liver tissues were obviously ameliorated by application of hUCB-MSCs and G-CSF. In conclusion, hUCB-MSCs, alone or co-treatment with G-CSF could ameliorate ALF in rats by inhibiting liver function injury, production of pro-inflammatory cytokines, oxidative stress, and liver cell apoptosis.

Preconditioning with interleukin-1 beta and interferon-gamma enhances the efficacy of human umbilical cord blood-derived mesenchymal stem cells-based therapy via enhancing prostaglandin E2 secretion and indoleamine 2,3-dioxygenase activity in dextran sulfate sodium-induced colitis.

Preconditioning with inflammatory cytokines has improved mesenchymal stem cells characteristics, including differentiation and immunomodulating functions. In this study, we developed a preconditioning combination strategy using interleukin-1beta (IL-1β) and interferon-gamma (IFN-γ) to enhance the immuneregulatory ability of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs). Our results showed that hUCB-MSCs preconditioned with IL-1β and IFN-γ (primed hUCB-MSCs) created a statistically significant decrease in peripheral blood mononuclear cell proliferation, indicating that their immunosuppressive ability was increased. The secretion of PGE2, cyclooxygenase 2 mRNA expression, and indoleamine 2,3-dioxygenase (IDO) mRNA expression in primed hUCB-MSCs was significantly higher than those in the untreated hUCB-MSCs or the IL-1β or IFN-γ only treated hUCB-MSCs. When inhibitors of IDO and PGE2 were treated, peripheral blood mononuclear cell proliferation, which is inhibited by primed hUCB-MSCs, was recovered. We found that Th1 T cell differentiation was also inhibited by PGE2 and IDO in the primed hUCB-MSCs, and Tregs differentiation was increased by PGE2 and IDO in the primed hUCB-MSCs. Furthermore, the primed hUCB-MSCs as well as supernatants increase CD4+ T cells migration. We demonstrated the therapeutic effects of primed hUCB-MSCs in dextran sulfate sodium-induced colitis model. In conclusion, we have demonstrated that primed hUCB-MSCs simultaneously enhance PGE2 and IDO and greatly improve the immunoregulatory capacity of MSCs, and we have developed an optimal condition for pretreatment of MSCs for the treatment of immune diseases. Our results raise the possibility that the combination of PGE2 and IDO could be therapeutic mediators for controlling immunosuppression of MSCs.

The Upregulation of Toll-Like Receptor 3 via Autocrine IFN-β Signaling Drives the Senescence of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Through JAK1.

Although mesenchymal stromal cells (MSCs) are among the most promising cell sources for cell-based therapies and regenerative medicine, the decline in their function with age due to cellular senescence limits their therapeutic applications. Unveiling the underlying mechanism of MSC senescence is therefore of substantial interest with regard to advancing MSC-based cell therapies. We here show that the induction of human umbilical cord blood-derived MSC (UCB-MSC) senescence causes the predominant upregulation of Toll-like receptor 3 (TLR3). Subsequent TLR3 activation by polyinosinic-polycytidylic acid triggers the prominent features of senescence. Using a clustered regularly interspaced short palindromic repeats/Cas9 library screening system, we identified Janus kinase 1 (JAK1) as the candidate regulatory factor for TLR3-mediated MSC senescence. A JAK1 deficiency blocked the MSC senescence phenotype upon TLR3 activation and TLR3 induction. Targeting the JAK1 pathway using chemical JAK1 inhibitors also significantly suppressed TLR3-mediated MSC senescence. Importantly, we further observed that UCB-MSC senescence is driven by a senescence-associated secretory phenotype (SASP) and that interferon-β (IFN-β) is a component of TLR3-dependent SASP, whereby its autocrine actions upregulate TLR3 and suppress cell proliferation. A JAK1 depletion significantly interrupted these effects of IFN-β, indicating that JAK1 is a signaling mediator linking IFN-β activity to TLR3 expression and the process of MSC senescence. Collectively, our findings provide new mechanistic insights into UCB-MSC senescence by revealing the role of an autocrine regulatory loop of SASP evoked by TLR3 activation.

Effects of hepatocyte growth factor gene-transfected mesenchymal stem cells on dimethylnitrosamine-induced liver fibrosis in rats

Nowadays, transplantation of human mesenchymal stem cells (MSCs) has emerged as a potential cellular therapy for liver cirrhosis. Hepatocyte growth factor (HGF) plays an important role in the regeneration of the liver. The objective of the study was to investigate the therapeutic effect of HGF-transfected human umbilical cord blood-derived MSCs on dimethylnitrosamine (DMN)-induced liver fibrosis in rats. HGF-transfected MSCs were transplanted into rats with DMN-induced liver fibrosis. H2O2-induced cytotoxicity, apoptosis and intracellular reactive oxygen species were reduced in HGF-transfected MSCs in HGF-transfected MSCs. Pro-apoptotic proteins, such as cleaved poly (ADP-ribose) polymerase and cleaved caspase-3, were decreased in HGF-transfected MSCs. Biochemical analysis showed that the levels of aspartate aminotransferase and alanine aminotransferase were decreased after transplantation of HGF-transfected MSCs in rat fibrosis. Trichrome staining showed that HGF-transfected MSCs reduced liver damage. Taken together, our study indicated that HGF-transfected MSCs have therapeutic effects on DMN-induced liver fibrosis in rats.

HUMAN UMBILICAL CORD BLOOD DERIVED MESENCHYMAL STEM CELLS REDUCE PHOSPHORYLATION AND AGGREGATION OF TAU IN AN ALZHEIMER’S DISEASE MODEL

In Alzheimer's Disease (AD), administration of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) mitigate hyperphosphorylation and aggregation of tau as well as improve impairment of spatial learning and memory in AD model. hUCB-MSCs were isolated and expanded from umbilical cord blood. The 5xFAD model rapidly develops severe amyloid pathology and appears cognitive impairment at 4 to 6 months-of-age. All assays were qualified or quantified by thioflavin T assay, immunofluorescence, western blot analysis, enzyme-linked immunosorbent assays and T-maze test. hUCB-MSCs have inhibitory effect on tau aggregation through in vitro aggregation assays using K18 fragment of human tau. Remarkably, hUCB-MSCs specifically decreases autophosphorylation of tyrosine 216 on the GSK-3β residue, as a kinase to promote phosphorylation of tau. Moreover, hUCB-MSCs effectively decreased phosphorylated forms of Tau at Thr181, Thr231 and Ser404, whereas effects of total Tau proteins on its stability by hUCB-MSCs were not detected. Strikingly, these phenomena mentioned above lead to results that paired helical filaments (PHF) of tau in the brain of 5XFAD mice diminished by treatment of hUCB-MSCs. Additionally, in T-maze testing, administered mice support that hUCB-MSCs have behavioral improvement of memory tasks most likely to occur in AD model. hUCB-MSCs diminish the phosphorylation and aggregation of tau through regulation of hUCB-MSCs, thus elucidating a therapeutic mechanism for AD.

Transplantation of stem cells from umbilical cord blood as therapy for type I diabetes.

In recent years, human umbilical cord blood has emerged as a rich source of stem, stromal and immune cells for cell-based therapy. Among the stem cells from umbilical cord blood, CD45+ multipotent stem cells and CD90+ mesenchymal stem cells have the potential to treat type I diabetes mellitus (T1DM), to correct autoimmune dysfunction and replenish β-cell numbers and function. In this review, we compare the general characteristics of umbilical cord blood-derived multipotent stem cells (UCB-SCs) and umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) and introduce their applications in T1DM. Although there are some differences in surface marker expression between UCB-SCs and UCB-MSCs, the two cell types display similar functions such as suppressing function of stimulated lymphocytes and imparting differentiation potential to insulin-producing cells (IPCs) in the setting of low immunogenicity, thereby providing a promising and safe approach for T1DM therapy.

Thrombin Preconditioning Boosts Biogenesis of Extracellular Vesicles from Mesenchymal Stem Cells and Enriches Their Cargo Contents via Protease-Activated Receptor-Mediated Signaling Pathways.

We investigated the role of protease-activated receptor (PAR)-mediated signaling pathways in the biogenesis of human umbilical cord blood-derived mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) and the enrichment of their cargo content after thrombin preconditioning. Immunoblot analyses showed that MSCs expressed two PAR subtypes: PAR-1 and PAR-3. Thrombin preconditioning significantly accelerated MSC-derived EV biogenesis more than five-fold and enriched their cargo contents by more than two-fold via activation of Rab5, early endosomal antigen (EEA)-1, and the extracellular signal regulated kinase (ERK)1/2 and AKT signaling pathways. Blockage of PAR-1 with the PAR-1-specific antagonist, SCH79797, significantly suppressed the activation of Rab5, EEA-1, and the ERK1/2 and AKT pathways and subsequently increased EV production and enriched EV cargo contents. Combined blockage of PAR-1 and PAR-3 further and significantly inhibited the activation of Rab5, EEA-1, and the ERK1/2 and AKT pathways, accelerated EV production, and enriched EV cargo contents. In summary, thrombin preconditioning boosted the biogenesis of MSC-derived EVs and enriched their cargo contents largely via PAR-1-mediated pathways and partly via PAR-1-independent, PAR-3-mediated activation of Rab5, EEA-1, and the ERK1/2 and AKT signaling pathways.

Traditional two-dimensional mesenchymal stem cells (MSCs) are better than spheroid MSCs on promoting retinal ganglion cells survival and axon regeneration

The loss of retinal ganglion cells (RGCs) is one of the common pathological features associated with optic nerve diseases leading to blindness. The aims of our study were to compare the neuroprotection of two forms of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) on RGCs and axon regeneration after optic nerve crush (ONC) in vivo, and to investigate the molecular mechanism. The effects of intravitreally transplanted hUCB-MSCs cultured in two-dimensional (2D-MSCs) and spheroids (3D-MSCs) were assessed by the survival of RGCs, regenerating axons, and flash visual evoked potentials (fVEP); the level of signal factors secreted by transplanted MSCs in vitreous and the marker protein levels of JAK/STAT3, PI3K/Akt/mTOR and MAPK/ERK pathways were detected using Bead-Based analysis and Western blot, respectively. We found that RGCs began to lose at day 3 after ONC, rapidly decreased at day 7, and flattened at day 14. The neuroprotection of transplanted 2D-MSCs was much stronger than that of 3D-MSCs. The transplanted 2D-MSCs could survive at least 2 weeks without differentiation and keep the characters of MSCs, which secreted multiple tropic factors and accompanied by activation of JAK/STAT3 and MAPK/ERK signaling pathways, top three most abundant factors: stem cell growth factor- β (SCGF-β), hepatocyte growth factor (HGF), and monocyte chemoattractant protein-1 (MCP-1). These results indicate that intravitreal injection of 2D-MSCs is a promising therapeutic strategy for retinal pathological diseases characterized by the loss of RGCs and open the door for the application of SCGF-β, HGF, and MCP-1 in the treatment of optic nerve diseases.

Mechanisms of the mixed umbilical cord blood-derived mesenchymal stem cells in inhibiting proliferation and inducing apoptosis of glioma cells

Objective To investigate the possible mechanism of mixed umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) in vitro to inhibit proliferation and induce apoptosis of C6 glioma cells. Methods The expression of B cell lymphoma/leukemia-2 (bcl-2) and cysteinyl aspartate-specific protease (Caspase)-3 proteins in C6 cells was detected by immunohistochemistry and Western blotting imprinting method, and C6 cell cycle was detected by flow cytometry. Results Different sources of UCB-MSCs have a tendency to increase the expression of apoptosis protein Caspase-3 in C6 cells and reduce the expression of apoptosis protein bcl-2. Different sources UCB-MSCs induce C6 cell apoptosis, E/T=0∶1, 5∶1, (2.5+ 2.5)∶1, 10∶1, (5+ 5)∶1 C6 cells acting on, The percentages of G0/G1 were (65.49±1.83)%, (64.83±2.19)%, (70.30±3.21)%, (69.03±2.19)%, (77.63±3.10)%, and the percentage of C6 cells in G0/G1 increased with the increase of the E/T ratio, showing a proportional dependence. Conclusion After phytohemagglutinin (PHA) stimulation, large doses of UCB-MSCs and mixed UCB-MSCs have inhibitory effects on proliferation of C6 cells in vitro, and this inhibition may be related to apoptosis induction Key words: Mesenchymal stem cells; Induce; Apoptosis

Interferon-γ-mediated secretion of tryptophanyl-tRNA synthetases has a role in protection of human umbilical cord blood-derived mesenchymal stem cells against experimental colitis

Mesenchymal stem cells (MSCs) are multipotent adult stem cells that present immunosuppressive effects in experimental and clinical trials targeting various rare diseases including inflammatory bowel disease (IBD). In addition, recent studies have reported tryptophanyl-tRNA synthetase (WRS) possesses uncanonical roles such as angiostatic and anti-inflammatory effects. However, little is known about the function of WRS in MSC-based therapy. In this study, we investigated if a novel factor, WRS, secreted from MSCs has a role in amelioration of IBD symptoms and determined a specific mechanism underlying MSC therapy. Experimental colitis was induced by administration of 3% DSS solution to 8-week-old mice and human umbilical cord blood-derived MSCs (hUCB-MSCs) were injected intraperitoneally. Secretion of WRS from hUCB-MSCs and direct effect of WRS on isolated CD4+ T cells was determined via in vitro experiments and hUCB-MSCs showed significant therapeutic rescue against experimental colitis. Importantly, WRS level in serum of colitis induced mice decreased and recovered by administration of MSCs. Through in vitro examination, WRS expression of hUCB-MSCs increased when cells were treated with interferon-γ (IFN-γ). WRS was evaluated and revealed to have a role in inhibiting activated T cells by inducing apoptosis. In summary, IFN-γ-mediated secretion of WRS from MSCs has a role in suppressive effect on excessive inflammation and disease progression of IBD and brings new highlights in the immunomodulatory potency of hUCB-MSCs.

PPIA, HPRT1, and YWHAZ Genes Are Suitable for Normalization of mRNA Expression in Long-Term Expanded Human Mesenchymal Stem Cells.

Long-term expansion of mesenchymal stem cells (MSCs) under defined culture conditions is necessary in human stem cell therapy. However, it alters the characteristics of MSCs. Since quantitative real time polymerase chain reaction (qRT-PCR) is widely used as one of the key analytical methods for comparative characterization, the validation of reference genes (RGs) for normalization under each experimental condition is important to achieve reliable qRT-PCR results. Therefore, the most stable RGs for long-term expanded bone marrow- and umbilical cord blood-derived MSCs (BM-MSCs and UCB-MSCs) under defined culture conditions for up to 20 passages were evaluated. The more apparent alterations in characteristics such as differentiation capacity, proliferation, senescence, and the expression of RGs were noted in BM-MSCs than UCB-MSCs during long-term expansion. The RG validation programs (GeNorm and NormFinder) suggested that PPIA, HPRT1, and YWHAZ were suitable for normalization in qRT-PCR regardless of MSC types and long-term culture expansion, and the traditional RGs (ACTB and GAPDH) were less stable in long-term expanded MSCs. In addition, the use of these RGs for normalization of OCT4 expression in long-term expanded BM-MSCs showed that a less stable RG (GAPDH) showed contrasting data compared to other RGs. Therefore, the use of RGs such as PPIA, HPRT1, and YWHAZ for normalization in qRT-PCR experiments is highly recommended for long-term expanded MSCs to generate accurate and reliable data.

Endothelin-1 Augments Therapeutic Potency of Human Mesenchymal Stem Cells via CDH2 and VEGF Signaling

In our previous study, we identified differences in the levels of CDH2 and vascular endothelial growth factor (VEGF) between effective and ineffective clones of human umbilical cord blood (hUCB) mesenchymal stem cells (MSCs), with regard to the infarcted rat myocardium. In this study, we compared gene expression profiles between the effective and ineffective clones and identified that endothelin-1 (EDN1) is enriched in the effective clone. In the mechanistic analyses, EDN1 significantly increased expression of CDH2 and VEGF through endothelin receptor A (EDNRA), which was prevented by EDNRA blocker, BQ123. To decipher how EDN1 induced gene expression of CDH2, we performed a promoter activity assay and identified GATA2 and MZF1 as inducers of CDH2. EDN1 significantly enhanced the promoter activity of the CDH2 gene, which was obliterated by the deletion or point mutation at GATA2 or MZF1 binding sequence. Next, therapeutic efficacy of EDN1-priming of hUCB-MSCs was tested in a rat myocardial infarction (MI) model. EDN1-primed MSCs were superior to naive MSCs at 8 weeks after MI in improving myocardial contractility (p < 0.05), reducing fibrosis area (p < 0.05), increasing engraftment efficiency (p < 0.05), and improving capillary density (p < 0.05). In conclusion, EDN1 induces CDH2 and VEGF expression in hUCB-MSCs, leading to the improved therapeutic efficacy in rat MI, suggesting that EDN1 is a potential priming agent for MSCs in regenerative medicine.

Exosomes Secreted by the Cocultures of Normal and Oxygen-Glucose-Deprived Stem Cells Improve Post-stroke Outcome.

Emerging stroke literature suggests that treatment of experimentally induced stroke with stem cells offered post-stroke neuroprotection via exosomes produced by these cells. Treatment with exosomes has great potential to overcome the limitations associated with cell-based therapies. However, in our preliminary studies, we noticed that the exosomes released from human umbilical cord blood-derived mesenchymal stem cells (MSCs) under standard culture conditions did not improve the post-stroke neurological outcome. Because of this apparent discrepancy, we hypothesized that exosome characteristics vary with the conditions of their production. Specifically, we suggest that the exosomes produced from the cocultures of regular and oxygen-glucose-deprived (OGD)MSCs in vitro would represent the exosomes produced from MSCs that are exposed to ischemic brain cells in vivo, and offer similar therapeutic benefits that the cell treatment would provide. We tested the efficacy of therapy with exosomes secreted from human umbilical cord blood(HUCB)-derived MSCs under in vitro hypoxic conditions on post-stroke brain damage and neurological outcome in a rat model of transient focal cerebral ischemia. We performed the TTC staining procedure as well as the neurological tests including the modified neurological severity scores(mNSS), the modified adhesive removal(sticky-tape), and the beam walking tests before ischemia and at regular intervals until 7 days reperfusion. Treatment with exosomes obtained from the cocultures of normal and OGD-induced MSCs reduced the infarct size and ipsilateral hemisphere swelling, preserved the neurological function, and facilitated the recovery of stroke-induced rats. Based on the results, we conclude that the treatment with exosomes secreted from MSCs at appropriate experimental conditions attenuates the post-stroke brain damage and improves the neurological outcome.

Mechanisms of mixed umbilical cord blood-derived mesenchymal stem cells inhibiting proliferation and inducing apoptosis of C6 glioma cells in vitro

Objective To study the mechanisms of the mixed umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) inhibiting proliferation and inducing apoptosis of C6 glioma cells in vitro. Methods Cysteinyl aspartate-specific protease (Caspase)-8, B cell lymphoma/leukemia-2 associated X protein (bax), B cell lymphoma/leukemia-2 (bcl-2) and Caspase-3 protein expression in C6 cells was analyzed by immunohistochemistry and Western blotting, respectively. Caspase-8, bax, bcl-2 and Caspase-3 mRNA levels were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Cell cycle was detected by flow cytometry. Apoptosis of C6 cells was examined by Annexin V-propidium iodide (PI) staining and terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) methods, respectively. Results The mixed UCB-MSCs had a tendency to increase the expression of Caspase-8, bax, Caspase-3 mRNA, and decrease the expression of bcl-2 mRNA. The expression of apoptosis protein and the number of apoptosis cells increased with the increase of the ratio of E/T (E: effect cells namely UCB-MSCs; T: target cells namely C6 cells), showing a proportional dependence relationship; E∶T=(5+ 5)∶1. The relative expression of Caspase-8, bax, bcl-2, and Caspase-3 mRNA in C6 cells was 0.78±0.13, 0.28±0.03, 0.70±0.08, and 0.76±0.12, respectively. When E/T=0∶1, 5∶1, (2.5+ 2.5)∶1, 10∶1, (5+ 5)∶1, the number of apoptosis C6 cells was (2.9±0.6), (3.1±0.5), (14.5±1.8), (14.3±2.2), and (22.8±4.5) cells, respectively. Conclusion After phytohemagglutinin (PHA) stimulation, the mixed UCB-MSCs could inhibit proliferation of C6 cells in vitro, which was related to induction of apoptosis of glioma cells. Key words: Glioma; Umbilical cord blood; Mesenchymal stem cells; Apoptosis; Proliferation