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Patients with refractory immune thrombocytopenia (ITP) frequently encounter substantial bleeding risks and demonstrate limited responsiveness to existing therapies. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) present a promising alternative, capitalizing on their low immunogenicity and potent immunomodulatory effects for treating diverse autoimmune disorders. This prospective phase I trial enrolled eighteen eligible patients to explore the safety and efficacy of UC-MSCs in treating refractory ITP. The research design included administering UC-MSCs at escalating doses of 0.5 × 10 6 cells/kg, 1.0 × 10 6 cells/kg, and 2.0 × 10 6 cells/kg weekly for four consecutive weeks across three cohorts during the dose-escalation phase, followed by a dose of 2.0 × 10 6 cells/kg weekly for the dose-expansion phase. Adverse events, platelet counts, and changes in peripheral blood immunity were monitored and recorded throughout the administration and follow-up period. Ultimately, 12 (with an addition of three patients in the 2.0 × 10 6 cells/kg group due to dose-limiting toxicity) and six patients were enrolled in the dose-escalation and dose-expansion phase, respectively. Thirteen patients (13/18, 72.2%) experienced one or more treatment emergent adverse events. Serious adverse events occurred in four patients (4/18, 22.2%), including gastrointestinal hemorrhage (2/4), profuse menstruation (1/4), and acute myocardial infarction (1/4). The response rates were 41.7% in the dose-escalation phase (5/12, two received 1.0 × 10 6 cells/kg per week, and three received 2.0 × 10 6 cells/kg per week) and 50.0% (3/6) in the dose-expansion phase. The overall response rate was 44.4% (8/18) among all enrolled patients. To sum up, UC-MSCs are effective and well tolerated in treating refractory ITP (ClinicalTrials.gov ID: NCT04014166)., (© 2024. The Author(s).)

Mesenchymal stem cells (MSCs) possess potent immunomodulatory activity and have been extensively investigated for their therapeutic potential in treating inflammatory disorders. However, the mechanisms underlying the immunosuppressive function of MSCs are not fully understood, hindering the development of standardized MSC-based therapies for clinical use. In this study, we profile the single-cell transcriptomes of MSCs isolated from adipose tissue (AD), bone marrow (BM), placental chorionic membrane (PM), and umbilical cord (UC). Our results demonstrate that MSCs undergo a progressive aging process and that the cellular senescence state influences their immunosuppressive activity by downregulating PD-L1 expression. Through integrated analysis of single-cell transcriptomic and proteomic data, we identify GATA2 as a regulator of MSC senescence and PD-L1 expression. Overall, our findings highlight the roles of cell aging and PD-L1 expression in modulating the immunosuppressive efficacy of MSCs and implicating perinatal MSC therapy for clinical applications in inflammatory disorders., (© 2023. The Author(s).)

Background: State-of-the-art advances have indicated the pivotal characteristics of bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) in hematopoietic microenvironment as well as coordinate contribution to hematological malignancies. However, the panoramic view and detailed dissection of BM-MSCs in patients with acute myeloid leukemia (AML-MSCs) remain obscure., Methods: For the purpose, we isolated and identified AML-MSCs together with healthy donor-derived HD-MSCs from the bone marrow mononuclear cells (BM-MNCs) by using the standard density gradient centrifugation based on clinical diagnosis and cellular phenotypic analysis. Subsequently, we systematically compared the potential similarities and discrepancy both at the cellular and molecular levels via flow cytometry, multilineage differentiation, chromosome karyotyping, cytokine quantification, and transcriptome sequencing and bioinformatic analysis including single-nucleotide polymorphism (SNP), gene ontology (GO), HeatMap, principal component analysis (PCA), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA)., Results: On the one hand, AML-MSCs exhibited undistinguishable signatures in cytomorphology, surface biomarker expression pattern, stemness, chromosome karyotype, and chondrogenesis as HD-MSCs, whereas with impaired adipogenesis, enhanced osteogenesis, and variations in cytokine expression pattern. On the other hand, with the aid of genomic and bioinformatic analyses, we verified that AML-MSCs displayed multidimensional discrepancy with HD-MSCs both in genome-wide gene expression profiling and genetic variation spectrum. Simultaneously, the deficiency of cellular vitality including proliferation and apoptosis in AML-MSCs was largely rescued by JAK-STAT signaling inhibition., Conclusions: Overall, our findings elucidated that AML-MSCs manifested multifaceted alterations in biological signatures and molecular genetics, and in particular, the deficiency of cellular vitality ascribed to over-activation of JAK-STAT signal, which collectively provided systematic and overwhelming new evidence for decoding the pathogenesis of AML and exploring therapeutic strategies in future.

Objective: Longitudinal studies have indicated VCAM-1 + mesenchymal stem/stromal cells (MSCs) as promising resources in regenerative medicine, yet the abundance in gene expression is far from adequate in the advantaged and "discarded" hUC-MSCs. Thus, high-efficient preparation and systematic dissection of the signatures and biofunctions of the subpopulation is the prerequisite for large-scale clinical applications., Materials and Methods: We primarily took advantage of a cytokine-based programming strategy for large-scale VCAM-1 + hUC-MSC generation (III-MSCs). Thereafter, we conducted multifaceted analyses including cytomorphology, immunophenotype, cell vitality, multilineage differentiation, whole-genome analysis, tube formation and Matrigel plug assay, lymphocyte activation and differentiation, and systemic transplantation for aplastic anaemia (AA) treatment., Results: III-MSCs with high-proportioned VCAM-1 expression were obtained by combining IL-1β, IL-4 with IFN-γ, which exhibited comparable immunophenotype with untreated hUC-MSCs (NT-MSCs) but revealed multidimensional superiorities both at the cellular and molecular levels. Simultaneously, systemic infusion of III-MSCs could significantly ameliorate clinicopathological features and finally help facilitate haematopoietic reconstruction and immunoregulation in AA mice., Conclusions: We have established a high-efficient procedure for large-scale generation of III-MSCs with preferable signatures and efficacy upon aplastic anaemia in mice. Our findings suggested that III-MSCs were advantageous sources with multifaceted characteristics for regenerative medicine., (© 2020 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd.)

Background: Mesenchymal stem cells are heterogenous populations with hematopoietic supporting and immunomodulating capacities. Enormous studies have focused on their preclinical or clinical therapeutic effects, yet the systematic study of continuous in vitro passages on signatures and functions of UC-MSCs at both the cellular and molecular levels is still lacking., Methods: In this study, to systematically evaluate the biological properties of MSCs at various passages, we analyzed biomarker expression, cell proliferation and apoptosis, chromosome karyotype, and tri-lineage differentiation potential. Subsequently, we took advantage of whole-exome sequencing to compare the somatic hypermutation of hUC-MSCs at P3, P6, and P15 including SNV and INDEL mutations. In addition, to explore the safety of the abovementioned hUC-MSCs, we performed metabolic pathway enrichment analysis and in vivo transplantation analysis. Furthermore, we cocultured the abovementioned hUC-MSCs with UCB-CD34 + HSCs to evaluate their hematopoietic supporting capacity in vitro. Finally, we transplanted the cells into acute graft-versus-host disease (aGVHD) mice to further evaluate their therapeutic effect in vivo., Results: The hUC-MSCs at P3, P6, and P15 showed similar morphology, biomarker expression, and cytokine secretion. hUC-MSCs at P15 had advantages on adipogenic differentiation and some cytokine secretion such as IL-6 and VEGF, with disadvantages on cell proliferation, apoptosis, and osteogenic and chondrogenic differentiation potential. Based on the SNP data of 334,378 exons and bioinformatic analyses, we found the somatic point mutations could be divided into 96 subsets and formed 30 kinds of signatures but did not show correlation with risk of tumorigenesis, which was confirmed by the in vivo transplantation experiments. However, hUC-MSCs at P15 showed impaired hematologic supporting effect in vitro and declined therapeutic effect on aGVHD in vivo., Conclusions: In this study, we systematically evaluated the biological and genetic properties of hUC-MSCs at various passages. Our findings have provided new references for safety and effectiveness assessments, which will provide overwhelming evidence for the safety of hUC-MSCs after continuous in vitro passages both at the cellular and molecular levels for the first time. Taken together, our studies could help understand the controversial effects of disease treatment and benefit the clinical research of UC-MSCs.

Background: Mesenchymal stem/stromal cells (MSCs) derived from human embryonic stem cells (hESCs) are attractive for their hematopoietic-supporting or potential therapeutic effects. However, procedures for high-effective and scalable generation of MSCs from hESCs within 2 weeks are still unestablished, which also hinder the development and mechanism study of mesengenesis., Methods: In this study, we aimed to establish a strategy for programming hESC differentiation into MSCs by practicing small-scale chemical compound screening. Then, we used flow cytometry, multi-lineage differentiation, and karyotype analyses to investigate the biological phenotypes of the derived hESC-MSCs. Also, to explore whether the derived cells had hematopoietic-supporting ability in vitro, we carried out the cobblestone formation and megakaryocytic differentiation experiments. To further evaluate the function of hESC-MSCs in vivo, we transplanted the cells into a mouse model with hind limb ischemia., Results: By simultaneous treatments with a JAK/STAT antagonist and a DNA methylation inhibitor, the efficiency of generating hESCs into CD73 + hESC-MPCs could reach 60% within 7 days. The derived cells further matured into hESC-MSCs, with comparable characteristics to those of adult MSCs in terms of surface markers, normal karyotype, and the potential for adipogenic, osteogenic, and chondrogenic differentiation. Functionally, hESC-MSCs had hematopoietic-supporting effects in vitro and could notably relieve symptoms of hind limb ischemia., Conclusions: In the study, we established a high-efficient procedure for large-scale generation of MSCs from hESCs, which would be of great help for genesis and mechanism studies of MSCs. Meanwhile, the derived cells provide an alternative for translational clinical research.

Objective: To investigate the effect of Deltex-1 on proliferation and differentiation of bone marrow mesenchymal stem cells (bMSCs) into smooth muscle cells (SMCs)., Materials and Methods: bMSCs of rat were isolated from bone marrow, cultured and identified. The effect of Deltex-1 on the proliferation of bMSCs infected with adenovirus vector pAd/Deltex-1 was detected by cell count kit-8 (CCK-8). The expression of smooth muscle myosin heavy chain (SM-MHC), that is one of the markers of SMCs in bMSCs without treatment, with Deltex-1 virus infection or empty virus infection and co-cultured with SMCs, were detected by immunofluorescence cytochemistry staining, RT-PCR and Western blotting. The same detection of bMSCs and SMCs without treatment was used as normal control, respectively., Results: bMSCs of rat were isolated from bone marrow, cultured and identified. Compared with the control, the results of CCK-8 showed that the growth of bMSCs infected by Deltex-1 virus was slower, and began to appear more significant especially at 48 (p<0.05, p<0.01). The results of immunofluorescence cytochemistry, Real-time PCR and Western blot showed that bMSCs with Deltex-1 virus infection and co-cultured with SMCs significantly expressed SM-MHC, and weakly expressed Notch-1., Conclusions: The proliferation of bMSCs with Deltex-1 over-expression could be inhibited and its differentiation into smooth muscle cells could be promoted.

Background Aims: Imatinib (IM), a tyrosine kinase inhibitor targeting the BCR-ABL oncoprotein, remains a major therapeutic strategy for patients with chronic myelogenous leukemia (CML). However, IM resistance is still a challenge in the treatment of CML. Recently, it was reported that exosomes (Exo) were involved in drug resistance. Therefore, the present study investigated whether Exo secreted by human umbilical cord mesenchymal stromal cells (hUC-MSC-Exo) affected the sensitivity of K562 cells to IM., Methods: hUC-MSC-Exo were isolated and identified. K562 cells were then treated or not with IM (1 µmol/L) in combination with hUC-MSC-Exo (50 µg/mL). Cell viability and apoptosis were determined by cell counting kit 8 (CCK-8) and annexin V/propidium iodide (PI) double staining, respectively. Apoptotic proteins, caspase and their cleaved forms were detected by Western blot., Results: It was shown that hUC-MSC-Exo alone had no effect on cell viability and apoptosis of K562 cells. However, hUC-MSC-Exo promoted IM-induced cell viability inhibition and apoptosis. Moreover, hUC-MSC-Exo enhanced the increased Bax expression and the decreased Bcl-2 expression that were induced by IM. Compared with IM alone, caspase-9 and caspase-3 were further activated by combination of hUC-MSC-Exo with IM. Finally, the effects of hUC-MSC-Exo on K562 cells could be reversed by pretreatment of K562 cells with caspase inhibitor Z-VAD-FMK (30 µmol/L) DISCUSSION: These results indicate that hUC-MSC-Exo enhanced the sensitivity of K562 cells to IM via activation of caspase signaling pathway. Therefore, combining IM with hUC-MSC-Exo could be a promising approach to improve the efficacy of CML treatment., (Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Mesenchymal stem cells (MSCs) have shown promise for use in cell therapy, and due to their tumor tropism can serve as vehicles for delivering therapeutic agents to tumor sites. Because interleukin-8 (IL-8) is known to mediate the protumor effect of MSCs, elimination of IL-8 secretion by MSCs may enhance their safety for use in cancer gene therapy. However, little is known concerning the effect of endogenously secreted IL-8 on MSCs. We performed studies using placenta-derived MSCs (PMSCs) to determine whether knockdown of IL-8 would influence their biological activity. We first verified that IL-8 and its membrane receptor CXCR2, but not CXCR1, were highly expressed in PMSCs. We then employed lentivirus-mediated small hairpin RNA interference to generate stable IL-8-silenced PMSCs, which displayed a variety of characteristic senescent phenotypes. We observed that at day 9 post-transfection, IL-8-silenced PMSCs had become larger and displayed a more flattened appearance when compared with their controls. Moreover, their proliferation, colony forming unit-fibroblast formation, adipogenic and osteogenic differentiation, and immunosuppressive potentials were significantly impaired. Enhanced senescence-associated β-galactosidase (SA-β-gal) activity and specific global gene expression profiles confirmed that IL-8 silencing evoked the senescence process in PMSCs. Increased levels of p-Akt and decreased levels of FOXO3a protein expression suggested that reactive oxygen species played a role in the initiation and maintenance of senescence in IL-8-silenced PMSCs. Notably, the majority of CXCR2 ligands were downregulated in presenescent IL-8-silenced PMSCs but upregulated in senescent cells, indicating an antagonistic pleiotropy of the IL-8/CXCR2 signaling pathway in PMSCs. This effect may promote the proliferation of young cells and accelerate senescence of old cells.

Background: Mori Fructus and Mori Ramulus are two traditional Chinese herbal medicines from mulberries. The present work explores their beneficial effects on •OH-treated mesenchymal stem cells (MSCs) and discusses possible mechanisms., Methods: Lyophilized aqueous extracts of Mori Fructus (LAMF) and Mori Ramulus (LAMR) were prepared and analyzed using HPLC. LAMF and LAMR (along with morin) were further investigated for their effects on •OH-treated MSCs using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay. The direct antioxidation mechanisms were studied using 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO•)-scavenging, 2,2'-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid (ABTS + •)-scavenging and 1,1-diphenyl-2-picryl-hydrazl (DPPH•)-scavenging, as well as Cu 2+ -reducing and Fe 3+ -reducing antioxidant power. Finally, the indirect antioxidant mechanism was investigated based on the UV-vis spectra of Fe 2+ -chelation., Results: In each LAMF and LAMR, seven phytophenols were successfully measured by HPLC, including five flavonoids (morin, rutin, astragalin, isoquercitrin and luteolin) and two non-flavonoids (chlorogenic acid and maclurin). MTT assays revealed that LAMF, LAMR and morin could effectively increase the survival of •OH-treated MSCs at 10-100 μg/mL, and could effectively scavenge PTIO• (IC 50 6609.7 ± 756.6, 4286.9 ± 84.9 and 103.4 ± 0.9 μg/mL, respectively), DPPH• (IC 50 208.7 ± 3.0, 97.3 ± 3.1 and 8.2 ± 0.7 μg/mL, respectively) and ABTS + • (IC 50 73.5 ± 5.8, 34.4 ± 0.1 and 4.2 ± 0.2 μg/mL, respectively), and reduce Cu 2+ (IC 50 212.5 ± 7.0, 123.2 ± 0.9 and 14.1 ± 0.04 μg/mL, respectively) & Fe 3+ (IC 50 277.0 ± 3.1, 191.9 ± 5.2 and 5.0 ± 0.2 μg/mL, respectively). In the Fe 2+ -chelating assay, the five flavonoids produced much stronger shoulder-peaks than the two non-flavonoids within 420-850 nm., Conclusion: Mori Fructus and Mori Ramulus, can protect MSCs from •OH-induced damage. Such beneficial effects can mainly be attributed to the antioxidant action of phytophenols, which occurs via direct (ROS-scavenging) and indirect mechanism (Fe 2+ -chelating). The ROS-scavenging mechanism, however, include at least a H + -transfer and an electron-transfer (ET), and possibly includes a hydrogen-atom-transfer (HAT). In the Fe 2+ -chelating, flavonoids are more effective than non-flavonoids. This can be attributed to several adjacent planar chelating-sites between the 3-OH and 4-C = O, between the 4-C = O and 5-OH, or between the 3'-OH and 4'-OH in flavonoids. Such multiple-Fe 2+ -chelating reactions cause overlap in the UV-vis absorptions to deepen the complex color, enhance the peak strength, and form shoulder-peaks. By comparison, two non-flavonoids with catechol moiety produce only a weak single peak.

Background: Mesenchymal stem cells (MSCs) have been widely proven effective for therapeutic angiogenesis in ischemia animal models as well as clinical vascular diseases. Because of the invasive method, limited resources, and aging problems of adult tissue-derived MSCs, more perinatal tissue-derived MSCs have been isolated and studied as promising substitutable MSCs for cell transplantation. However, fewer studies have comparatively studied the angiogenic efficacy of MSCs derived from different tissues sources. Here, we evaluated whether the in-situ environment would affect the angiogenic potential of MSCs., Methods: We harvested MSCs from adult bone marrow (BMSCs), adipose tissue (AMSCs), perinatal umbilical cord (UMSCs), and placental chorionic villi (PMSCs), and studied their "MSC identity" by flow cytometry and in-vitro trilineage differentiation assay. Then we comparatively studied their endothelial differentiation capabilities and paracrine actions side by side in vitro., Results: Our data showed that UMSCs and PMSCs fitted well with the minimum standard of MSCs as well as BMSCs and AMSCs. Interestingly, we found that MSCs regardless of their tissue origins could develop similar endothelial-relevant functions in vitro, including producing eNOS and uptaking ac-LDL during endothelial differentiation in spite of their feeble expression of endothelial-related genes and proteins. Additionally, we surprisingly found that BMSCs and PMSCs could directly form tubular structures in vitro on Matrigel and their conditioned medium showed significant proangiogenic bioactivities on endothelial cells in vitro compared with those of AMSCs and UMSCs. Besides, several angiogenic genes were upregulated in BMSCs and PMSCs in comparison with AMSCs and UMSCs. Moreover, enzyme-linked immunosorbent assay further confirmed that BMSCs secreted much more VEGF, and PMSCs secreted much more HGF and PGE2., Conclusions: Our study demonstrated the heterogeneous proangiogenic properties of MSCs derived from different tissue origins, and the in vivo isolated environment might contribute to these differences. Our study suggested that MSCs derived from bone marrow and placental chorionic villi might be preferred in clinical application for therapeutic angiogenesis.

Numerous studies have demonstrated that interferon-γ (IFN-γ) is an important inflammatory cytokine, which may activate the immunomodulatory abilities of mesenchymal stem cells (MSCs), and may influence certain other functions of these cells. MicroRNAs are small non‑coding RNAs that regulate the majority of the biological functions of cells and are important in a variety of biological processes. However, few studies have been performed to investigate whether IFN‑γ affects the microRNA profile of MSCs. The aim of the present study was to analyze the microRNA profile of MSCs derived from the umbilical cord (UC‑MSCs) cultured in the presence or absence of IFN‑γ (IFN‑UC‑MSCs). An array that detects 754 microRNAs was used to determine the expression profiles. Statistical analysis of the array data revealed that 8 microRNAs were significantly differentially expressed in UC‑MSCs and IFN‑UC‑MSCs. Reverse transcription‑quantitative polymerase chain reaction validated the differential expression of the 8 identified microRNAs. The target genes of the 8 microRNAs were predicted through two online databases, TargetScan and miRanda, and the predicted results were screened by bioinformatics analysis. The majority of the target genes were involved in the regulation of transcription, signal transduction, proliferation, differentiation and migration. These results may provide insight into the mechanism underlying the regulation of the biological functions of MSCs by IFN‑γ, in particular the immunomodulatory activity.

Objective: To investigate the effect of microRNA-382 (miR-382) on the biological properties of human umbilical cord-derived mesenchymal stem cells (hUC-MSC)., Methods: The mimics and inhibitor of miR-382 were transfected into hUC-MSC with lipo2000. Inverted microscopy was used to observe the morphology change of hUC-MSC. The proliferation of hUC-MSC was detected by CCK-8. Oil red O and alizarin red staining were applied to assess the adipogenic and osteogenic differentiation of hUC-MSC. Cetylpyridinium chloride was used to the quantitative analysis of osteogenic differentiation. The expression of Runx2 and some cytokines were detected by RT-PCR., Results: miR-382 did not influence the morphology, proliferation and adipogenic differentiation of hUC-MSC miR-382 inhibited the expression of Runx2, thus could inhibit the osteogenesis of hUC-MSC, being confirmed by alizarin red stain; miR-382 could influence the expression of key cytokines secreted from hUC-MSC, such as IL-6, IDO1, G-CSF, M-CSF, GM-CSF., Conclusion: miR-382 decreases the expression of Runx2 and inhibites the osteogenesis of hUC-MSC. In addition, it also affects the expression of some key cytokines secreted from hUC-MSC.

Introduction: Mesenchymal stem cells (MSCs) represent a heterogeneous cell population that is promising for regenerative medicine. The present study was designed to assess whether VCAM-1 can be used as a marker of MSC subpopulation with superior angiogenic potential., Methods: MSCs were isolated from placenta chorionic villi (CV). The VCAM-1(+/-) CV-MSCs population were separated by Flow Cytometry and subjected to a comparative analysis for their angiogenic properties including angiogenic genes expression, vasculo-angiogenic abilities on Matrigel in vitro and in vivo, angiogenic paracrine activities, cytokine array, and therapeutic angiogenesis in vascular ischemic diseases., Results: Angiogenic genes, including HGF, ANG, IL8, IL6, VEGF-A, TGFβ, MMP2 and bFGF, were up-regulated in VCAM-1(+)CV-MSCs. Consistently, angiogenic cytokines especially HGF, IL8, angiogenin, angiopoitin-2, μPAR, CXCL1, IL-1β, IL-1α, CSF2, CSF3, MCP-3, CTACK, and OPG were found to be significantly increased in VCAM-1(+) CV-MSCs. Moreover, VCAM-1(+)CV-MSCs showed remarkable vasculo-angiogenic abilities by angiogenesis analysis with Matrigel in vitro and in vivo and the conditioned medium of VCAM-1(+) CV-MSCs exerted markedly pro-proliferative and pro-migratory effects on endothelial cells compared to VCAM-1(-)CV-MSCs. Finally, transplantation of VCAM-1(+)CV-MSCs into the ischemic hind limb of BALB/c nude mice resulted in a significantly functional improvement in comparison with VCAM-1(-)CV-MSCs transplantation., Conclusions: VCAM-1(+)CV-MSCs possessed a favorable angiogenic paracrine activity and displayed therapeutic efficacy on hindlimb ischemia. Our results suggested that VCAM-1(+)CV-MSCs may represent an important subpopulation of MSC for efficient therapeutic angiogenesis.

Objective: To investigate the effect of human umbilical cord-derived mesenchymal stem cells (UC-MSC) on the differentiation of leukemic cells., Methods: The co-culture system of UC-MSC with acute promyelocytic leukemic cell line NB4 cells was constructed in vitro,and the differentiation status of the leukemic cells was assessed by cell morphology,nitroblue tetrazolium reduction test,and cell surface differentiation marker CD11b., Results: UC-MSC induced the granulocytic differentiation of NB4 cells. When UC-MSC and a small dose of all-trans retinoic acid were applied together,the differentiation-inducing effect was enhanced in an additive manner. Interleukin (IL)-6Ra neutralization attenuated differentiation and exogenous IL-6-induced differentiation of leukemic cells., Conclusion: UC-MSC can promotd granulocytic differentiation of acute promyelocytic leukemia cells by way of IL-6 and presented additive effect when combined with a small dose of all-trans retinoic acid.

Promising results are emerging in clinical trials focused on stem cell therapy for cardiology applications. However, the low homing and engraftment of the injected cells to target tissue continues to be a problem. Cellular glycoengineering can address this limitation by enabling the targeting of stem cells to sites of vascular injury/inflammation. Two such glycoengineering methods are presented here: i. The non-covalent incorporation of a P-selectin glycoprotein ligand-1 (PSGL-1) mimetic 19Fc[FUT7(+)] via lipid-protein G fusion intermediates that intercalate onto the cell surface, and ii. Over-expression of the α(1,3)fucosyltransferse FUT7 in cells. Results demonstrate the efficient coupling of 19Fc[FUT7(+)] onto both cardiosphere-derived cells (CDCs) and mesenchymal stem cells (MSCs), with coupling being more efficient when using protein G fused to single-tailed palmitic acid rather than double-tailed DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine). This non-covalent cellular modification was mild since cell proliferation and stem-cell marker expression was unaltered. Whereas coupling using 19Fc[FUT7(+)] enhanced cell capture on recombinant P-selectin or CHO-P cell surfaces, α(1,3)fucosylation was necessary for robust binding to E-selectin and inflamed endothelial cells under shear. Pilot studies confirm the safety and homing efficacy of the modified stem cells to sites of ischemia-reperfusion in the porcine heart. Overall, glycoengineering with physiological selectin-ligands may enhance stem cell engraftment., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Although emerging evidence links mesenchymal stem cells (MSCs) with cancer metastasis, the underlying mechanisms are poorly understood. In the present study, we found that human umbilical cord-derived MSCs (UC-MSCs) promoted MCF-7 cell migration in vitro and metastasis in vivo. To explore the mechanisms, the characteristics of MCF-7 cells cocultured with UC-MSCs were assessed. The expression and secretion of interleukin-8 (IL-8) and IL-6 were induced in MCF-7 cells cocultured with UC-MSCs. However, neutralization of IL-8 or IL-6 secreted by UC-MSCs could attenuate the enhanced expression of IL-8 and IL-6 in MCF-7 cells cocultured with UC-MSCs, which subsequently alleviated the enhanced migration. Similar to UC-MSCs, exogenous human recombinant IL-8 or IL-6 also promoted IL-8 and IL-6 expression and MCF-7 cell migration. In addition to enhanced IL-8 and IL-6 expression, MCF-7 cells cocultured with UC-MSCs displayed enhanced mammosphere-forming ability and increased percentage of CD44(+)/CD24(-) cells. However, epithelial-to-mesenchymal transition (EMT) was not observed in MCF-7 cells cocultured with UC-MSCs. Taken together, these results suggested that IL-8 and IL-6 secreted by UC-MSCs activated the autocrine IL-8 and IL-6 signaling in MCF-7 cells and induced CD44(+)/CD24(-) cells, which subsequently promoted MCF-7 cell migration in vitro and metastasis in vivo.

Background: The Notch signaling pathway is implicated in a broad range of developmental processes, including cell fate decisions. This study was designed to determine the role of Notch signaling in adipogenic differentiation of human bone marrow derived MSCs (BM-MSCs)., Methods: The Notch signaling was inhibited by the γ-secretase inhibitor N-[N-(3,5-difluor- ophenacetyl-L-alanyl)]-S-phenylglycine t-butylester (DAPT). The markers involving adipogenic differentiation of MSCs, the relative pathway PTEN-PI3K/Akt/mTOR and autophagy activation were then analyzed. Furthermore, the autophagy inhibitor chloroquine (CQ) and 3-methyladenine (3-MA) were used to study the role of autophagy in the DAPT-induced the adipogenic differentiation of MSCs., Results: We first confirmed the down -regulation of Notch gene expression during MSCs adipocyte differentiation, and showed that the inhibition of Notch signaling significantly enhanced adipogenic differentiation of MSCs. Furthermore, Notch inhibitor DAPT induced early autophagy by acting on PTEN-PI3K/Akt/mTOR pathway. The autophagy inhibitor CQ and 3-MA dramatically abolished the effects of DAPT-induced autophagy and adipogenic differentiation of MSCs., Conclusion: Our results indicate that inhibition of Notch signaling could promote MSCs adipogenesis mediated by autophagy involving PTEN-PI3K/Akt/mTOR pathway. Notch signaling could be a novel target for regulating the adipogenic differentiation of MSCs., (© 2015 S. Karger AG, Basel.)

Introduction: Mesenchymal stem cells (MSCs) are widely investigated in clinical researches to treat various diseases. Classic culture medium for MSCs, even for clinical use, contains fetal bovine serum. The serum-containing medium (SCM) seems a major obstacle for MSCs-related therapies due to the risk of contamination of infectious pathogens. Some studies showed that MSCs could be expanded in serum free medium (SFM); however, whether SFM would change the biological characteristics and safety issues of MSCs has not been well answered., Methods: Human umbilical cord mesenchymal stem cells (hUC-MSCs) were cultured in a chemical defined serum free medium. Growth, multipotency, surface antigen expression, telomerase, immunosuppressive ability, gene expression profile and genomic stability of hUC-MSCs cultured in SFM and SCM were analyzed and compared side by side., Results: hUC-MSCs propagated more slowly and senesce ultimately in SFM. SFM-expanded hUC-MSCs were different from SCM-expanded hUC-MSCs in growth rate, telomerase, gene expression profile. However, SFM-expanded hUC-MSCs maintained multipotency and the profile of surface antigen which were used to define human MSCs. Both SFM- and SCM-expanded hUC-MSCs gained copy number variation (CNV) in long-term in vitro culture., Conclusion: hUC-MCSs could be expanded in SFM safely to obtain enough cells for clinical application, meeting the basic criteria for human mesenchymal stem cells. hUC-MSCs cultured in SFM were distinct from hUC-MSCs cultured in SCM, yet they remained therapeutic potentials for future regenerative medicine.

Chronic immune thrombocytopenia (ITP) is characterized by autoimmune-mediated platelet destruction and impairment of thrombopoiesis. Mesenchymal stem cells (MSCs) are proposed to exhibit immune modulatory functions in self-tolerance maintenance. In this study, we aimed to characterize phenotypically and functionally bone marrow (BM)-derived MSCs from adult chronic ITP patients. Our results showed that BM-MSCs from patients with chronic ITP exhibited impaired proliferation, abnormal morphology and excessive apoptosis, and these defects could be ameliorated by modifying the culture environment. BM-MSCs from chronic ITP patients were shown to have similar immunophenotype and capacities to differentiate along adipogenic and osteogenic lineages as those from normal controls. However, the immune-inhibiting potential and the regulatory T cell-inducing ability of BM-MSCs from patients were defective compared to that of normal BM-MSCs. These findings suggest that the BM-MSCs were defective in chronic ITP patients. Whether the defective BM-MSCs are relevant to the pathogenesis of chronic ITP remains to be determined.

This study aimed to investigate the significance of cytokine expression in supernatant from hematopoietic stem/progenitor cells (HSCs/HPCs) co-cultured with mesenchymal stem cells (MSCs) or endothelial progenitor cells (EPCs). Mononuclear cells (MNCs) were isolated from normal human umbilical cord blood and then cultured solely or co-cultured with MSCs or EPCs. Changes in the number of MNCs and HSCs/HPCs were observed, and MNC proliferation was tested by carboxyfluorescein diacetate succinimidyl ester. The cultured supernatants of the treated MSCs and EPCs were collected at 24 h after co-culture and used to determine the concentrations of IL-3, IL-6, stem cell factor (SCF), TPO, Flt3l, and VEGF. The total number and proliferation of MNCs increased significantly when co-cultured with MSCs or EPCs than when cultured alone, particularly when MNCs were co-cultured with EPCs. The differences in IL-3 and Flt3l concentrations between groups were not significant. However, IL-6 in the MSC group was significantly higher than that in the two other groups. The SCF and TPO concentrations were highly expressed in the EPC group. The VEGF concentrations in the MSC group and the EPC group were higher than those in the control group. These results indicated that MSCs and EPCs possibly favor the proliferation of MNCs and HSCs/HPCs. IL-6 and VEGF may be related to hematopoietic reconstitution and homing ability of HSCs/HPCs. TPO may have a specific relationship with the promotion of HSCs/HPCs differentiation.

This study was aimed to explore the immunoregulatory function and capability supporting the angiogenesis of exosomes secreted by bone marrow mesenchymal stem cells (BMMSC) from healthy persons. Supernatant of BMMSC (P4-P6) was collected for exosome purification. Transmission electron microscopy (TEM) and Western blot were used to identify the quality of isolated exosomes. The amount of exosomes was quantified through bicinchoninic acid (BCA) protein assay. Human peripheral blood mononuclear cells (PBMNC) were isolated from healthy donor and added with isolating exosomes. After co-cultured for 72 h, IFN-γ from the co-culture system was detected by ELISA. The expression of miRNA-associated with immunity were detected by real-time reverse transcription polymerase chain reaction (Real-time RT-PCR). The interactions between exosomes and human umbilical vein endothelial cells (HUVEC) were observed with confocal microscopy. Subconfluent HUVEC were harvested and treated with the indicated concentration of exosomes. Nude mice were injected subcutaneously with exosomes or PBS as control to verify the ability of angiogenesis. The results showed that diameter range of exosomes was range from 40 to 160 nm. The isolated exosomes expressed the CD9. There was approximately linear relation between the secretion of exosomes and cell density. The exosomes suppressed the production of IFN-γ from PBMNC, and contained miRNA associated with immune regulation such as miR301, miR22 and miR-let-7a. Exosomes induced vascular tube formation in vitro and vascularization of Matrigel plugs in vivo. It is concluded that the BMMSC-derived exosomes can regulate immunity and support vascularization.

Mesenchymal stem cells (MSCs) could be obtained from many sources, and there are differences between them. This study was purposed to compare and analyze the basic biological characteristics of umbilical cord, adipose tissue-and bone marrow-derived MSC (UC-MSCs, AD-MSCs and BM-MSCs). The MSCs were isolated from umbilical cord, adipose tissue and bone marrow were cultured; the morphology of UC-MSCs, AD-MSCs and BM-MSCs was observed by using microscopy; the immunophenotype, differentiation potential and expression of peroxisome proliferation-activated receptor-γ (PPAR-γ) mRNA were detected by using flow cytometry, differentiation test (von kossais and 0:1 red O staining) and quantitative fluorescent PCR, respectively. The results showed that the UC-MSCs, AD-MSCs and BM-MSCs displayed similar morphology under confocal microscope after being stained with rhodamine phalloidin and DAPL. The immunophenotypes of these three originated cells conform to coincide with identification criterion for MSCs, and showed similar expression level. During adipogenic induction the adipogenic potential of these MSCs was different, AD-MSCs exhibited the highest adipogenic potential, UC-MSCs displayed the lowest, while potential of BM-MSCs get between; however, the osteogenic differentiation potential of UC-MSCs, AD-MSCs and BM-MSCs was similar. The PCR detection showed that the expression level of PPAR-γ mRNA was the highest in AD-MSCs and the lowest in UC-MSCs, while expression level in BM-MSCs get between, these results were identical with the adipogenic potential, suggest that the difference of adipogenic potential in 3 kinds of MSCs was associated with basic expression level of PPAR-γ mRNA. It is concluded that UC-MSCs, AD-MSCs and BM-MSCs exhibit similar morphology, the immunophenotypes of these MSCs coincide with identification criterion for MSCs, the osteogenic potential of these MSCs is similar, while the adipogenic potential and the expression level of PPAR-γ mRNA are different. The difference-associated mechanisms need to further study.

This study was aimed to investigate the effects of rapamycin on biological function and autophagy of bone marrow mesenchymal stem cells (BM-MSC) from patients with aplastic anemia so as to provide experimental basis for the clinical treatment of aplastic anemia (AA) with rapamycin. BM-MSC were treated with different concentrations of rapamycin (0, 10, 50, 100 nmol/L) for 48 h, the expression of LC3B protein was detected by Western blot to observe the effect of rapamycin on cell autophagy; cell apoptosis and cell cycles were detected by flow cytometry; the proliferation of BM-MSC of AA patients was measured by cell counting kit-8; the adipogenic differentiation of BM-MSC were tested by oil red O staining after adipogenic induction for 2 weeks; the adipogenic related genes (LPL, CFD, PPARγ) were detected by real-time PCR. The results showed that the proliferation and adipogenesis of BM-MSC of AA patients were inhibited by rapamycin. Moreover, the autophagy and apoptosis of BM-MSC were increased by rapamycin in a dose-dependent way.Rapamycin arrested the BM-MSC in G0/G1 phase and prevented them into S phase (P < 0.05). It is concluded that rapamycin plays an critical role in inhibiting cell proliferation, cell cycles, and adipogenesis, these effects may be related with the autophagy activation and mTOR inhibition resulting from rapamycin.

Cultured human umbilical cord mesenchymal stem cells (hUC-MSCs) are being tested in several clinical trials and encouraging outcomes have been observed. To determine whether in vitro expansion influences the genomic stability of hUC-MSCs, we maintained nine hUC-MSC clones in long-term culture and comparatively analyzed them at early and late passages. All of the clones senesced in culture, exhibiting decreased telomerase activity and shortened telomeres. Two clones showed no DNA copy number variations (CNVs) at passage 30 (P30). Seven clones had ≥1 CNVs at P30 compared with P3, and one of these clones appeared trisomic chromosome 10 at the late passage. No tumor developed in immunodeficient mice injected with hUC-MSCs, regardless of whether the cells had CNVs at the late passage. mRNA-Seq analysis indicated that pathways of cell cycle control and DNA damage response were downregulated during in vitro culture in hUC-MSC clones that showed genomic instability, but the same pathways were upregulated in the clones with good genomic stability. These results demonstrated that hUC-MSCs can be cultured for many passages and attain a large number of cells, but most of the cultured hUC-MSCs develop genomic alterations. Although hUC-MSCs with genomic alterations do not undergo malignant transformation, periodic genomic monitoring and donor management focusing on genomic stability are recommended before these cells are used for clinical applications.

15-Deoxy-Δ(12), 14-prostaglandin J2 (15d-PGJ2), a well known peroxisome proliferator activated receptor (PPAR) γ ligand, has been shown to inhibit cellular proliferation and induce apoptosis and differentiation. However, whether 15d-PGJ2 influences the cytokines in the culture supernatant of bone marrow mesenchymal stem cells (BM-MSC) is unknown. This study was purposed to investigate the influence of 15d-PGJ2 on cytokines in the culture supernatant of BM-MSC. The fibroblast-like cells attached to the culture dish from bone marrow of healthy donors were isolated. The immunophenotype and differentiation potential of the obtained cells were detected by flow cytometry and oil red O and von kassa staining respectively to confirm that these cells were BM-MSC. Thereafter, the BM-MSC were cultured with complete medium supplemented with 10, 20, 40 and 60 µmol/L 15d-PGJ2 for 24 hours respectively. The real-time PCR was used to assay the PPARγ mRNA level, the confocal immuno fluorescence technique was used to detect the expression level of PPARγ. The results showed that the BM-MSC underwent apoptosis and got detached from the culture dish when the concentration of 15d-PGJ2 was no less than 20 µmol/L. The PPARγ mRNA level of BM-MSCs cultured with medium containing 10 µmol/L 15d-PGJ2 was higher than that cultured without 15d-PGJ2, and the difference was statistically significant (P < 0.05). The enhancement of PPARγ expression was observed after stimulated by 15d-PGJ2. The protein chip detecting the culture supernatants of BM-MSC cultured with 10 µmol/L 15d-PGJ2 or without 15d-PGJ2 for 24 hours demonstrated that expression levels of some of the cytokines varied. It is concluded that the down-regulation of TIMP-2 exists after treatment of 15d-PGJ2, which is statistical significant.

Background Aims: Toll-like receptors (TLRs) are expressed in human umbilical cord-derived mesenchymal stromal cells (UC-MSCs), and activation of TLRs plays an important role in proliferation, differentiation and immunoregulatory activity of UC-MSCs. We investigated whether TLRs regulated the expression of microRNAs (miRNAs) in UC-MSCs and the role of miRNAs., Methods and Results: With miRNA microarray analysis, we demonstrated that the expression of many miRNAs varied when UC-MSCs were stimulated with the ligand of toll-like receptor 4 (TLR4), lipopolysaccharide (LPS). The expression of some miRNAs was verified by polymerase chain reaction. It was found that microRNA-301a (miR-301a) was up-regulated by the ligands of TLR3 and TLR4, LPS and polyinosinic acid:polycytidylic acid poly(I:C). However, the inhibitors of nuclear factor κB NF-κB and interferon regulatory factor 3 IRF3 signal attenuated the effect of LPS and poly(I:C) on miR-301a expression. Over-expression or lower expression of miR-301a affected the cytokine secretion of UC-MSCs., Conclusions: The expression of miR-301a in UC-MSCs was regulated by TLRs, and miR-301a affected the cytokine secretion of UC-MSCs., (Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Mesenchymal stem/stromal cells (MSCs) are an important candidate for cell-based therapy since they can be easily isolated and expanded, secrete beneficial paracrine factors, and differentiate into multiple lineages. Since the endothelium at sites of injury and inflammation often express adhesion molecules belonging to the selectin family, methods to endow MSCs with selectin-ligands can enhance the efficacy of cell delivery and tissue engraftment. Here, we describe a construct 19Fc[FUT7(+)], where the first 19 amino acids of the pan-selectin ligand PSGL-1 (P-selectin glycoprotein ligand-1) was fused to a human IgG1. When expressed in HEK293T cells over-expressing the α(1,3)fucosyltransferase FUT7, 19Fc[FUT7(+)] is decorated by a core-2 sialyl Lewis-X sialofucosylated O-glycan. The non-covalent coupling of this protein onto MSC surface using palmitated protein G (PPG) enhanced cell binding to E- and P-selectin under hydrodynamic shear, without altering MSC multipotency. MSCs functionalized with 19Fc[FUT7(+)] were captured/tethered onto stimulated endothelial cell monolayers at wall shear stresses up to 4 dyn/cm(2). Once captured, the cells rolled robustly up to the highest shear stress tested, 10 dyn/cm(2). Unlike previous work where MSCs could only be captured onto selectin-bearing substrates at low or no-flow conditions, the current work presents a 'glycan engineering' strategy to enable leukocyte-like capture and rolling., (© 2013 Elsevier Ltd. All rights reserved.)

This study was aimed to investigate the effect of IL-1β on hematopoietic support of human umbilical cord mesenchymal stem cells (hUC-MSC). 2×10(6) hUC-MSC were seeded in 75 cm(2) flasks, after adherence to wall for 2 h, 10 ng/ml IL-1β was added in hUC-MSC supernatant and cultured for 36 h, then the culture supernatants and cells were harvested. The effect of conditioned medium with/without IL-1β on CD34(+) cell hematopoietic support was observed, mRNA expression changes of hUC-MSC cultured in medium with/without IL-1β were monitored by real time PCR, the differences in hematopoiesis-related factors were detected by ELISA. The results showed that the conditioned culture medium of hUC-MSC with IL-1β enhanced the ability to form colony of CD34(+) cells, especially CFU-G and CFU-GM in vitro; IL-1β promoted the mRNA expression of GM-CSF, G-CSF, IL-6 on MSC; IL-1β also promoted the secretion of GM-CSF, G-CSF, and IL-6 protein from hUC-MSC. It is concluded that IL-1β enhances hematopoietic support capacity especially, capability of MSC to myeloid differentiation.

All-trans retinoic acid (ATRA) induces clinical remission in most acute promyelocytic leukemia (APL) patients by inducing terminal differentiation of APL cells toward mature granulocytes. Here we report that human umbilical cord-derived mesenchymal stem cells (UC-MSCs) are capable of inducing granulocytic differentiation of the APL-derived NB4 cell line as well as primary APL cells and also cooperate with ATRA in an additive manner. Transwell coculture experiments revealed that UC-MSCs' differentiation-inducing effect was mediated through some soluble factors. Differentiation attenuation by IL-6Ra neutralization and induction by addition of exogenous IL-6 confirmed that IL-6 secreted by UC-MSCs was at least partially responsible for this differentiation induction process. Moreover, we found that UC-MSCs activated the MEK/ERK signaling pathway in promyelocytic cells and pharmacological inhibition of the MEK/ERK pathway reversed UC-MSC-induced differentiation, indicating that UC-MSCs exerted effect through activation of the MEK/ERK signaling pathway. These results demonstrate for the first time a stimulatory effect of MSCs on the differentiation of APL cells and bring a new insight into the interaction between MSCs and leukemic cells. Our data suggest that UC-MSCs/ATRA combination could be used as a novel therapeutic strategy for APL patients.

This study was purposed to investigate the impact of human umbilical cord-derived mesenchymal stem cells (hUC-MSC) on the sensitivity of HL-60 cells to therapeutic drugs so as to provide more information for exploring the regulatory effect of hUC-MSC on leukemia cells. Transwell and direct co-culture systems of HL-60 and hUC-MSC were established. The apoptosis and cell cycle of HL-60 cells were detected by flow cytometry. RT-PCR and Western blot were used to detect the mRNA and protein levels of Caspase 3, respectively. The results showed that the apoptosis of HL-60 induced by cytarabine (Ara-C) decreased significantly after direct co-cultured with hUC-MSC cycle mRNA (P < 0.05). The similar phenomenon was observed in transwell co-culture system. Cell cycle of HL-60 cells were arrested at G0/G1 phase and did not enter into S phase (P < 0.05) and the expression of Caspase-3 mRNA and protein in HL-60 cells were reduced (P < 0.05). It is concluded that hUC-MSC protected HL-60 from Arc-C induced apoptosis through regulating the cell cycle and down-regulating expression of Caspase 3 in HL-60 cells. In addition, this effect is caused by the soluble factors from hUC-MSC.

Comparing to bone marrow mesenchymal stem cells (MSCs), placenta-derived MSCs have the advantages of adequate sources, low immunogenicity, little risk of viral contamination, and no ethical controversy, and thus possess a better prospect for clinical application. Placental tissue not only includes chorionic and amniotic, but also contains decidua basalis which locate in the maternal placenta surface. The biological characteristics of MSCs isolated from decidua basalis have not been well studied. This study was aimed to investigate the biologic characteristics of placenta decidua basalis-derived MSC from placenta decidua basalis (DB) by enzymatic digestion. Short tandem repeats (STR) test was used to identify the cells derived from the maternal placenta surface. Growth rate of decidua basalis mesenchymal stem cells (DB-MSC) was measured by MTT. Cell cycle and cell phenotype were detected by flow cytometry. Inducing differentiation was used to evaluate multipotency of DB-MSC. For testing the immunosuppression of DB-MSC, they were co-cultured with peripheral blood mononuclear cells (PBMNC) stimulated by phytohemagglutinin (PHA) and then IFN-γ in the co-cultured media was quantified by ELISA. The results showed that the cells were derived from the maternal placenta by STR analysis. DB-MSC showed typical fibroblast morphology in the culture and were positive for the MSC surface markers: CD90, CD73, CD105, CD44 and negative for CD45, CD11b, and CD34. DB-MSC underwent osteogenic, adipogenic and chondrogenic differentiation in inducing medium. DB-MSC could inhibit the secretion of IFN-γ by PBMNC. It is concluded that the cells are isolated from placenta decidua basalis and possess the basic characteristics of MSC. DB-MSC can be an important maternal autologous MSC and may be a safe and effective treatment for immune system diseases, which makes the DB-MSC as an important source of autologous MSC from mother. DB-MSC can be safely for the treatment of the mother's immune system diseases.

Background Aims: Toll-like receptors (TLRs) play an important role in innate and adaptive immunity by recognizing pathogen-associated molecular patterns (PAMPs)., Methods: In the present study, we investigated the expression and role of TLRs on human umbilical cord mesenchymal stromal cells (UC-MSCs). The proliferation, differentiation and immunoregulatory activity of UC-MSCs primed with or without TLR ligands were determined., Results: At the RNA level, the expression of TLR2, 4, 6 and 9 was relatively higher than that of other TLRs. However, TLR3 and TLR4 expression were relatively higher at the protein level. UC-MSCs expressed functional TLRs by nuclear factor-κB activation and cytokine expression assay. Poly-inosinic acid:cytidylic acid [Poly(I:C)] stimulation inhibited the proliferation of UC-MSCs, but the ligand of other TLRs had no significant effect. Poly(I:C) stimulation enhanced the adipogenic differentiation capability of UC-MSCs, but lipopolysaccharide inhibited the adipogenic differentiation. Poly(I:C) and CpG-oligonucleotide promoted the immunosuppressive potentiality of UC-MSCs, accompanied with the phosphorylation of interferon regulatory factor 3 (IRF3) and increased expression of indoleamine 2,3-dioxygenase and interferon β, whereas activation of other TLR ligands (synthetic analog fibroblast-stimulating lipopeptide-1 and lipopolysaccharide) failed to affect the immunoregulatory activity of UC-MSCs., Conclusions: Taken together, our data demonstrated that TLR activation influenced the function of UC-MSCs, which might have important implications in future efforts to explore the clinical potentials of UC-MSCs., (Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Mesenchymal stem cells (MSCs) reside in almost all of the body tissues, where they undergo self-renewal and multi-lineage differentiation. MSCs derived from different tissues share many similarities but also show some differences in term of biological properties. We aim to search for significant differences among various sources of MSCs and to explore their implications in physiopathology and clinical translation. We compared the phenotype and biological properties among different MSCs isolated from human term placental chorionic villi (CV), umbilical cord (UC), adult bone marrow (BM) and adipose (AD). We found that CD106 (VCAM-1) was expressed highest on the CV-MSCs, moderately on BM-MSCs, lightly on UC-MSCs and absent on AD-MSCs. CV-MSCs also showed unique immune-associated gene expression and immunomodulation. We thus separated CD106(+)cells and CD106(-)cells from CV-MSCs and compared their biological activities. Both two subpopulations were capable of osteogenic and adipogenic differentiation while CD106(+)CV-MSCs were more effective to modulate T helper subsets but possessed decreased colony formation capacity. In addition, CD106(+)CV-MSCs expressed more cytokines than CD106(-)CV-MSCs. These data demonstrate that CD106 identifies a subpopulation of CV-MSCs with unique immunoregulatory activity and reveal a previously unrecognized mechanism underlying immunomodulation of MSCs.

Because advantage of tissue origin and proliferation potential, the umbilical cord-derived mesenchymal stem cells (UC-MSC) and placental chorionic villous-derived mesenchymal stem cells (CV-MSC) have clinical application potential, as compared with bone marrow MSC. But whether the differences of biological characteristics exist between UC-MSC and CV-MSC, which deserve to be further explored. This study was purposed to compare the biological characteristics of UC-MSC and CV-MSC. The placental and umbilical cord were cleaned by using the sterile physiological salt, the UC-MSC and CV-MSC were separated by enzyme digestion. Short tandem repeat (STR) analysis was used to detect whether the MSC obtained from fetal tissue. MTT method was used to detect proliferation of MSC. Flow cytometry was applied to analyze cell phenotype. The different differential medium was used to detect their multi-directional differentiation capacity. After the MSC and PHA-stimulated peripheral blood mononuclear cells were co-cultured, the γ-interferon (IFN-γ) levels of the co-culture supernatant were detected using the ELISA. The results showed that these MSC were derived from fetal tissue by STR analysis. They were adherent cells with typical fibroblast morphology. Cells expressed the MSC surface markers CD90, CD73 and CD105 and CD44, not expressed CD45 and of CD11b and CD34.These cells could differentiate into osteoblasts and adipoblasts under culture with different conditioned medium, but in the adipogenic differentiation of CV-MSC, the larger lipid droplets appeared. It is concluded that these cells are obtained MSC. These MSC can inhibit peripheral blood mononuclear cells stimulated by PHA to secrete IFN-γ, and the the CV-MSC have a stronger suppression capacity, which makes the CV-MSC to have a greater advantage in the treatment of autoimmune diseases.

The aim of this study was to investigate the effects of interferon (IFN)-γ on biological characteristics and immunomodulatory property of human umbilical cord-derived mesenchymal stem cells (hUC-MSC). hUC-MSC were treated with IFN-γ 10 ng/ml (IFN-γ group) or without IFN-γ (control group). The phenotype of hUC-MSC was detected by flow cytometry. The proliferation status was detected by CCK-8 method, and its differentiation ability was assessed by oil red O and von Kossa staining. The production of PGE-2 was measured by ELISA, and the mRNA expression levels of COX-2, IDO-1 and IDO-2 in hUC-MSC were detected by real-time quantitative PCR. Furthermore, the proliferation of human peripheral blood mononuclear cells (hPBMNC) was evaluated after co-culture with hUC-MSC, IFN-γ pretreatment or not. The results showed that after IFN-γ stimulation, the expression of SSEA-4 on hUC-MSC decreased significantly [(8.15 ± 2.94) vs (16.42 ± 8.5), P < 0.05], and the expression of CD54 increased [(96.64 ± 3.29) vs (84.12 ± 10.73), P = 0.051]. The immunomodulatory property of hUC-MSC on the proliferation of hPBMNC was enhanced (P < 0.05). All the above mentioned effects were IFN-γ concentration-dependent. When hUC-MSC were stimulated by IFN-γ for 24 h, the production of PGE-2 secreted by hUC-MSC decreased significantly (P < 0.01). The mRNA expression level of COX-2 also decreased though the difference did not reach to statistically significant level. Compared with control group, IDO-1 expression level in IFN-γ group increased significantly (P < 0.01), and the mRNA expression level of IDO-2 remained unchanged. It is concluded that IFN-γ can influence the phenotype of hUC-MSC and enhance the immunomodulatory property of hUC-MSC.

Background/aims: Human umbilical cord mesenchymal stem cells (hUC-MSCs) possess immunosuppressive activities but the mechanisms of such activities are not fully understood. Here, we investigated the role of IL-6, one of the characteristic factors of MSCs, in the immunoregulating effect of hUC-MSCs on CD4(+) T lymphocytes., Methods: The condition media from human peripheral blood mononuclear cells (hPBMCs) or CD14+/- cell were tested if stimulating IL-6 production by hUC-MSCs. The related signaling pathway of IL-6, and the immunosuppressive activity of IL-6 on CD4(+) T lymphocytes were studied., Result: IL-6 production was dramatically increased by hUC-MSCs when co-culturing with resting or activated hPBMCs. CD14(+) monocytes-paracrined IL-1β promoted the secretion of IL-6 by hUC-MSCs via JNK and NF-κB signaling pathway. Blocking of PGE2 synthesis did not affect the secretion of IL-6, anti-IL-6 antibody was not able to reverse hUC-MSCs-mediated inhibition on CD4(+) T lymphocytes. IL-6 did not mediate the suppressive activity of IL-1β-hUC-MSCs- PGE2 on CD4(+) T cell., Conclusion: CD14(+) monocytes-paracrined IL-1β promotes IL-6 secretion by hUC-MSCs through activating JNK and NF-κB signaling pathway. However, increased IL-6 production does not contribute to immunosuppressive activity of IL-1β-hUC-MSCs- PGE2 on CD4(+) T cells., (Copyright © 2012 S. Karger AG, Basel.)

Background: Mesenchymal stem cells (MSC) play important roles in modulating the activities of T lymphocytes, dendritic cells and natural killer cells. These immunoregulatory properties of MSC suggest their therapeutic potential in autoimmune diseases. However, the effects of MSC on B cells are still poorly understood. The present study was designed to investigate the interaction between MSC and B cells both in vitro and in vivo, and to determine the possible mechanism of action., Design and Method: The effect of human umbilical cord mesenchymal stem cells (UC-MSC) on proliferation and differentiation of B-cells were characterized in vitro, and we also tested the immunoregulatory properties of mouse bone marrow MSC (BM-MSC) on T cell dependent and independent antibody production in vivo in mice., Results: Treatment with human UC-MSC resulted in an increase of proliferation, differentiation of B cells into plasma cells and production of antibodies in vitro. Mouse BM-MSC significantly enhanced T cell dependent and independent antibodies production in vivo in mice. PGE2 partially mediated the immunosuppressive activity of human UC-MSC but IL-6 did not regulate this activity., Conclusion: MSC promote proliferation and differentiation of B cells in vitro and in vivo partially through PGE2 but not IL-6., (Copyright © 2012 S. Karger AG, Basel.)

Hematopoietic stem cells (HSC) shift from fetal liver and spleen to bone marrow at neonatal stages and this movement may be due to inductive signals from different microenvironments. Mesenchymal stem cells (MSC) are the precursors of stromal cells in bone marrow microenvironments such as osteoblasts and endothelial cells. Some researchers speculated that fetal bone marrow before birth might be not perfectly suit HSC growth. However, it is still lack of direct evidence to prove this hypothesis. This study was aimed to compare the hematopoietic supportive capacity between human fetal and adult bone marrow MSC in vitro. Adult bone marrow MSC (ABM-MSC) were isolated from three healthy donors and fetal bone marrow MSC (FBM-MSC) were isolated from three fetuses between gestations of 19 to 20 weeks. After irradiation, MSC were co-cultured with CD34(+) cells isolated from umbilical cord blood in long-term culture-initiating cell (LTC-IC) assay. The colony number of colony forming cells (CFC) was counted and the phenotypic changes of co-cultured CD34(+) cells were analyzed by flow cytometry. Cytokine expressions in both kinds of MSC were detected by reverse transcription polymerase chain reaction (RT-PCR). The results showed that ABM-MSC had a stronger hematopoietic supportive capacity than FBM-MSC. Both of them enhanced the differentiation of CD34(+) cells into myeloid lineages. Cytokines were expressed differently in ABM-MSC and FBM-MSC. It is concluded that ABM-MSC possess more potential application in some treatments than FBM-MSC, especially in hematopoietic reconstitution.

Here, the effect of CD14(+) monocytes on human umbilical cord matrix stem cell (hUC-MSC)-mediated immunosuppression was studied in vitro. hUC-MSCs exerted a potent inhibitory effect on the proliferation and interferon-gamma (IFN-gamma) secretion capacities of CD4(+) and CD8(+) T cells in response to anti-CD3/CD28 stimulation. Transwell co-culture system revealed that the suppressive effect was primarily mediated by soluble factors. Addition of prostaglandin synthesis inhibitors (indomethacin or NS-398) almost completely abrogated the immunosuppression activity of hUC-MSCs, identifying prostaglandin E(2) (PGE(2)) as an important soluble mediator. CD14(+) monocytes were found to be able to enhance significantly the immunosuppressive effect of hUC-MSCs in a dose-dependent fashion. Moreover, the inflammatory cytokine IL-1beta, either exogenously added or produced by CD14(+) monocytes in culture, could trigger expression of high levels of PGE(2) by hUC-MSCs, whereas inclusion of the IL-1 receptor antagonist (IL-1RA) in the culture down-regulated not only PGE(2) expression, but also reversed the promotional effect of CD14(+) monocytes and partially restored CD4(+) and CD8(+) T cell proliferation and IFN-gamma secretion. Our data demonstrate an important role of monocytes in the hUC-MSC-induced immunomodulation, which may have important implications in future efforts to explore the clinical potentials of hUC-MSCs., (Copyright 2010 Elsevier Inc. All rights reserved.)

In real-time quantitative PCR, the accuracy of normalized data is highly dependent on the stability of the reference genes. However, reference gene expression in a given cell type or experimental condition can vary considerably. The goal of this study was to establish a reliable set of reference genes for real-time PCR studies using human umbilical cord mesenchymal stem cells with long-term in vitro expansion. The stability of ten potential reference genes was examined in human umbilical cord mesenchymal stem cells. We found that Ywhaz and Rpl13a, not beta-actin or Gapdh, were the most stably expressed of the internal control genes in different passages of human umbilical cord mesenchymal stem cells. Ywhaz and Rpl13a could be used as reference genes for relative gene quantification and normalization purposes in real-time PCR studies of human umbilical cord mesenchymal stem cells.

Human umbilical-cord-derived mesenchymal stem cells (hUC-MSCs) constitute an attractive alternative to bone-marrow-derived MSCs for potential clinical applications because of easy preparation and lower risk of viral contamination. In this study, both proliferation of human peripheral blood mononuclear cells (hPBMCs) and their IFN-gamma production in response to mitogenic or allogeneic stimulus were effectively inhibited by hUC-MSCs. Co-culture experiments in transwell systems indicated that the suppression was largely mediated by soluble factor(s). Blocking experiments identified prostaglandin E(2) (PGE(2)) as the major factor, because inhibition of PGE(2) synthesis almost completely mitigated the immunosuppressive effects, whereas neutralization of TGF-beta, IDO, and NO activities had little effects. Moreover, the inflammatory cytokines, IFN-gamma and IL-1beta, produced by hPBMCs upon activation notably upregulated the expression of cyclooxygenase-2 (COX-2) and the production of PGE(2) by hUC-MSCs. In conclusion, our data have demonstrated for the first time the PGE(2)-mediated mechanism by which hUC-MSCs exert their immunomodulatory effects., (Copyright 2010 Elsevier Inc. All rights reserved.)

Background Aims: The acceleration of capillarization and venularization of hepatic sinusoids after cell therapy would not be beneficial to restoration after liver disease. The goal was to observe the effects of umbilical cord (UC)-derived mesenchymal stromal cells (MSC) on liver microcirculation and their therapeutic potential in liver fibrosis., Methods: Human UC MSC labeled with or without CM-DIL were transplanted into NOD/SCID mice with carbon tetrachloride (CCl4)-induced chronic liver fibrosis models. Because of the high autofluorescence on the injured liver sections, we used immunohistochemistry, Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR), but not immunofluorescence, in order to avoid false images under a confocal fluorescence microscope., Results: Human-specific alpha-fetoprotein and albumin mRNA and proteins were detected in CCl4-treated mouse livers receiving human UC MSC transplants. We only observed the gene expression of human-specific endothelial-like cells markers CD31 and KDR by RT-PCR, but not protein expression by immunohistochemistry, in UC MSC-transplanted mouse livers. Vascular endothelial growth factor (VEGF) expression in injured livers 4 weeks after UC MSC transplantation was higher than in normal livers. However, UC MSC injection did not increase significantly the vascular density labeled by CD31 and (vWF) in the injured livers of UC MSC-transplanted mice compared with non-transplanted mice after CCl4 treatment. In addition, liver function was partly improved after UC MSC transplantation., Conclusions: Human UC MSC can differentiate into hepatocyte-like cells but do not accelerate the capillarization and venularization of hepatic sinusoids, finally leading to the partial improvement of liver function in mice with CCl4-mediated chronic liver fibrosis.

Senescence of bone marrow mesenchymal stem cells (BMMSCs) induced by chronic oxidative stress is an important factor contributes to the postmenopausal osteoporosis (PMOP). Mitochondrial quality control takes a pivotal role in regulating oxidative stress and cell senescence. Genistein is a major isoflavone in soy products, which is best known for its ability to inhibit bone loss in both postmenopausal women and ovariectomized (OVX) rodents. Here we show that OVX-BMMSCs displayed premature senescence, elevated reactive oxygen species (ROS) level and mitochondria dysfunction, while genistein rescued these phenotypes. Using network pharmacology and molecular docking, we identified estrogen-related receptor α (ERRα) as the potential target of genistein. Knockdown of ERRα greatly abolished the anti-senescence effect of genistein on OVX-BMMSCs. Further, the mitochondrial biogenesis and mitophagy induced by genistein were inhibited by ERRα knockdown in OVX-BMMSCs. In vivo, genistein inhibited trabecular bone loss and p16INK4a expression, upregulated sirtuin 3 (SIRT3) and peroxisome proliferator-activated receptor gamma coactivator one alpha (PGC1α) expression in the trabecular bone area of proximal tibia in OVX rats. Together, this study revealed that genistein ameliorates senescence of OVX-BMMSCs through ERRα-mediated mitochondrial biogenesis and mitophagy, which provided a molecular basis for advancement and development of therapeutic strategies against PMOP.

Abstract Background Dentigerous cyst (DC) is a bone destructive disease and remains a challenge for clinicians. Marsupialization enables the bone to regenerate with capsule maintaining, making it a preferred therapeutic means for DC adjacent to vital anatomical structures. Given that capsules of DC are derived from odontogenic epithelium remnants at the embryonic stage, we investigated whether there were mesenchymal stem cells (MSCs) located in DC capsules and the role that they played in the bone regeneration after marsupialization. Methods Samples obtained before and after marsupialization were used for histological detection and cell culture. The stemness of cells isolated from fresh tissues was analyzed by morphology, surface marker, and multi-differentiation assays. Comparison of proliferation ability between MSCs isolated from DC capsules before (Bm-DCSCs) and after (Am-DCSCs) marsupialization was evaluated by Cell Counting Kit-8 (CCK-8), fibroblast colony-forming units (CFU-F), and 5′-ethynyl-2′-deoxyuridine (EdU) assay. Their osteogenic capacity in vitro was detected by alkaline phosphatase (ALP) and Alizarin Red staining (ARS), combined with real-time polymerase chain reaction (RT-PCR) and immunofluorescence (IF) staining. Subcutaneous ectopic osteogenesis as well as cranial bone defect model in nude mice was performed to detect their bone regeneration and bone defect repairability. Results Bone tissue and strong ALP activity were detected in the capsule of DC after marsupialization. Two types of MSCs were isolated from fibrous capsules of DC both before (Bm-DCSCs) and after (Am-DCSCs) marsupialization. These fibroblast-like, colony-forming cells expressed MSC markers (CD44+, CD90+, CD31−, CD34−, CD45−), and they could differentiate into osteoblast-, adipocyte-, and chondrocyte-like cells under induction. Notably, Am-DCSCs performed better in cell proliferation and self-renewal. Moreover, Am-DCSCs showed a greater osteogenic capacity both in vitro and in vivo compared with Bm-DCSCs. Conclusions There are MSCs residing in capsules of DC, and the cell viability as well as the osteogenic capacity of them is largely enhanced after marsupialization. Our findings suggested that MSCs might play a crucial role in the healing process of DC after marsupialization, thus providing new insight into the treatment for DC by promoting the osteogenic differentiation of MSCs inside capsules.

Expression of E-cadherin, a hallmark of epithelial-mesenchymal transition (EMT), is often lost due to promoter DNA methylation in basal-like breast cancer (BLBC), which contributes to the metastatic advantage of this disease; however, the underlying mechanism remains unclear. Here, we identified that Snail interacted with Suv39H1 (suppressor of variegation 3-9 homolog 1), a major methyltransferase responsible for H3K9me3 that intimately links to DNA methylation. We demonstrated that the SNAG domain of Snail and the SET domain of Suv39H1 were required for their mutual interactions. We found that H3K9me3 and DNA methylation on the E-cadherin promoter were higher in BLBC cell lines. We showed that Snail interacted with Suv39H1 and recruited it to the E-cadherin promoter for transcriptional repression. Knockdown of Suv39H1 restored E-cadherin expression by blocking H3K9me3 and DNA methylation and resulted in the inhibition of cell migration, invasion and metastasis of BLBC. Our study not only reveals a critical mechanism underlying the epigenetic regulation of EMT, but also paves a way for the development of new treatment strategies against this disease. [ABSTRACT FROM AUTHOR]