Internalization of nanopolymeric tracers does not alter characteristics of placental cells

In the cell therapy scenario, efficient tracing of transplanted cells is essential for investigating cell migration and interactions with host tissues. This is fundamental to provide mechanistic insights which altogether allow for the understanding of the translational potential of placental cell therapy in the clinical setting. Mesenchymal stem/stromal cells (MSC) from human placenta are increasingly being investigated for their potential in treating patients with a variety of diseases. In this study, we investigated the feasibility of using poly (methyl methacrylate) nanoparticles (PMMA‐NPs) to trace placental MSC, namely those from the amniotic membrane (hAMSC) and early chorionic villi (hCV‐MSC). We report that PMMP‐NPs are efficiently internalized and retained in both populations, and do not alter cell morphofunctional parameters. We observed that PMMP‐NP incorporation does not alter in vitro immune modulatory capability of placental MSC, a characteristic central to their reparative/therapeutic effects in vitro. We also show that in vitro, PMMP‐NP uptake is not affected by hypoxia. Interestingly, after in vivo brain ischaemia and reperfusion injury achieved by transient middle cerebral artery occlusion (tMCAo) in mice, iv hAMSC treatment resulted in significant improvement in cognitive function compared to PBS‐treated tMCAo mice. Our study provides evidence that tracing placental MSC with PMMP‐NPs does not alter their in vitro and in vivo functions. These observations are grounds for the use of PMMP‐NPs as tools to investigate the therapeutic mechanisms of hAMSC and hCV‐MSC in preclinical models of inflammatory‐driven diseases.     

Adipose-derived stromal cells inhibit prostate cancer cell proliferation inducing apoptosis

Mesenchymal stem cells (MSCs) have generated a great deal of interest in the field of regenerative medicine. Adipose-derived stromal cells (AdSCs) are known to exhibit extensive proliferation potential and can undergo multilineage differentiation, sharing similar characteristics to bone marrow-derived MSCs. However, as the effect of AdSCs on tumor growth has not been studied sufficiently, we assessed the degree to which AdSCs affect the proliferation of prostate cancer (PCa) cell. Human AdSCs exerted an inhibitory effect on the proliferation of androgen-responsive (LNCaP) and androgen-nonresponsive (PC3) human PCa cells, while normal human dermal fibroblasts (NHDFs) did not, and in fact promoted PCa cell proliferation to a degree. Moreover, AdSCs induced apoptosis of LNCaP cells and PC3 cells, activating the caspase3/7 signaling pathway. cDNA microarray analysis suggested that AdSC-induced apoptosis in both LNCaP and PC3 cells was related to the TGF-β signaling pathway. Consistent with our in vitro observations, local transplantation of AdSCs delayed the growth of tumors derived from both LNCaP- and PC3-xenografts in immunodeficient mice. This is the first preclinical study to have directly demonstrated that AdSC-induced PCa cell apoptosis may occur via the TGF-β signaling pathway, irrespective of androgen-responsiveness. Since autologous AdSCs can be easily isolated from adipose tissue without any ethical concerns, we suggest that therapy with these cells could be a novel approach for patients with PCa.     

Antiproliferative mechanism of a cannabinoid agonist by cell cycle arrest in human gastric cancer cells

For gastric cancers, the antineoplastic activity of cannabinoids has been investigated in only a few reports and knowledge regarding the mechanisms involved is limited. We have reported previously that treatment of gastric cancer cells with a cannabinoid agonist significantly decreased cell proliferation and induced apoptosis. Here, we evaluated the effects of cannabinoids on various cellular mediators involved in cell cycle arrest in gastric cancer cells. AGS and MKN-1 cell lines were used as human gastric cancer cells and WIN 55,212-2 as a cannabinoid agonist. Cell cycles were analyzed by flow cytometry and western blotting. Treatment with WIN 55,212-2 arrested the cell cycle in the G0/G1 phase. WIN 55,212-2 also upregulated phospho-ERK1/2, induced Kip1/p27 and Cip1/WAF1/p21 expression, decreased cyclin D1 and cyclin E expression, decreased Cdk 2, Cdk 4, and Cdk 6 expression levels, and decreased phospho-Rb and E2F-1 expression. ERK inhibitor decreased the proportion of G0/G1 phase which was induced by WIN 55,212-2. Inhibition of pAKT led to cell cycle arrest in gastric cancer cells. Cell cycle arrest preceded apoptotic response. Thus, this cannabinoid agonist can reduce gastric cancer cell proliferation via G1 phase cell cycle arrest, which is mediated via activation of the MAPK pathway and inhibition of pAKT.     

Altered expression of G1/S phase cell cycle regulators in placental mesenchymal stromal cells derived from preeclamptic pregnancies with fetal-placental compromise

Herein, we evaluated whether Placental Mesenchymal Stromal Cells (PDMSCs) derived from normal and Preeclamptic (PE) placentae presented differences in the expression of G1/S-phase regulators p16^INK4A, p18^INK4C, CDK4 and CDK6. Finally, we investigated normal and PE-PDMSCs paracrine effects on JunB, Cyclin D1, p16^INK4A, p18^INK4C, CDK4 and CDK6 expressions in physiological term villous explants.

PDMSCs were isolated from physiological (n = 20) and PE (n = 24) placentae. Passage three normal and PE-PDMSC and conditioned media (CM) were collected after 48h. Physiological villous explants (n = 60) were treated for 72h with normal or PE-PDMSCs CM. Explants viability was assessed by Lactate Dehydrogenase Cytotoxicity assay. Cyclin D1 localization was evaluated by Immuofluorescence (IF) while JunB, Cyclin-D1 p16^INK4A, p18^INK4C, CDK4 and CDK6 levels were assessed by Real Time PCR and Western Blot assay.

We reported significantly increased p16^INK4A and p18^INK4C expression in PE- relative to normal PDMSCs while no differences in CDK4 and CDK6 levels were detected. Explants viability was not affected by normal or PE-PDMSCs CM. Normal PDMSCs CM increased JunB, p16^INK4 and p18^INK4C and decreased Cyclin-D1 in placental tissues. In contrast, PE-PDMSCs CM induced JunB downregulation and Cyclin D1 increase in placental explants. Cyclin D1 IF staining showed that CM treatment targeted mainly the syncytiotrophoblast.

We showed Cyclin D1-p16INK4A/p18INK4C altered pathway in PE-PDMSCs demonstrating an aberrant G1/S phase transition in these pathological cells. The abnormal Cyclin D1-p16INK4A/p18INK4C expression in explants conditioned by PE-PDMSCs media suggest a key contribution of mesenchymal cells to the altered trophoblast cell cycle regulation typical of PE pregnancies with fetal-placental compromise.     

Microenvironment mesenchymal cells protect ovarian cancer cell lines from apoptosis by inhibiting XIAP inactivation

Epithelial ovarian carcinoma is characterized by high frequency of recurrence (70% of patients) and carboplatin resistance acquisition. Carcinoma-associated mesenchymal stem cells (CA-MSC) have been shown to induce ovarian cancer chemoresistance through trogocytosis. Here we examined CA-MSC properties to protect ovarian cancer cells from carboplatin-induced apoptosis. Apoptosis was determined by Propidium Iodide and Annexin-V-FITC labelling and poly-ADP-ribose polymerase cleavage analysis. We showed a significant increase of inhibitory concentration 50 and a 30% decrease of carboplatin-induced apoptosis in ovarian cancer cells incubated in the presence of CA-MSC-conditioned medium (CM). A molecular analysis of apoptosis signalling pathway in response to carboplatin revealed that the presence of CA-MSC CM induced a 30% decrease of effector caspases-3 and -7 activation and proteolysis activity. CA-MSC secretions promoted Akt and X-linked inhibitor of apoptosis protein (XIAP; caspase inhibitor from inhibitor of apoptosis protein (IAP) family) phosphorylation. XIAP depletion by siRNA strategy permitted to restore apoptosis in ovarian cancer cells stimulated by CA-MSC CM. The factors secreted by CA-MSC are able to confer chemoresistance to carboplatin in ovarian cancer cells through the inhibition of effector caspases activation and apoptosis blockade. Activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway and the phosphorylation of its downstream target XIAP underlined the implication of this signalling pathway in ovarian cancer chemoresistance. This study reveals the potentialities of targeting XIAP in ovarian cancer therapy.     

二甲双胍对人脐带间充质干细胞形态、增殖、表面标志及细胞周期的影响

目的探讨不同浓度二甲双胍（METF）对人脐带间充质干细胞（hUC-MSC）形态、增殖、表面标志及细胞周期的影响。 方法取健康足月新生儿脐带在体外分离出hUC-MSC进行传代培养,至第3代（流式细胞仪分析）对细胞进行鉴定,取第6代处于对数生长期的hUC-MSC （相对老化）,将对照组与不同浓度METF （0.1,1,5,10,20?mmol/L）干预的细胞进行比较,观察不同浓度METF干预对细胞的形态、增殖率（MTT法分别于24、48、72?h检测）、及细胞表面标志和细胞周期的影响,采用One-Way ANOVA,及LSD-t检验进行统计学分析。 结果（1）METF为0.1?mmol/L、1?mmol/L,细胞形态无显著改变,当药物浓度为5?~?20?mmol/?L时,随着药物浓度增加、培养时间延长,细胞形态改变越显著。（2）METF为0.1?mmol/L（24?h：101.28±0.98,24?h：104.06±1.76,24?h：101.51±0.67）促进hUC-MSC增殖,药物浓度为1?~ 10?mmol/L在培养初期可增加间充质干细胞的增殖率,随着培养时间的延长,细胞的增殖逐渐被抑制。METF为20?mmol/L（24?h：86.64±0.66,48?h：58.38±2.52,72?h：17.75±1.35）抑制细胞增殖,抑制作用随着时间延长而增强（P?< 0.05）。（3）当METF浓度为5,10,20?mmol/L时,随着药物浓度的增加,CD105的表达逐渐减弱（F?= 17.539,P?< 0.05）。METF未对CD44、CD90产生影响。（4）METF为0.1?mmol/L时降低G0/G1期的比例（64.16±1.20,P?< 0.05）,促进间充质干细胞的增殖,随着药物浓度的增加,细胞增殖逐渐被抑制。 结论METF浓度在0.1mmol/?L促进hUC-MSC增殖,而在浓度5 ~ 20?mmol/L时抑制人脐带间充质干细胞的增殖及表面标志CD105的表达,不同浓度的METF均未对CD44、CD90的表达产生影响。     

Co-culture with mesenchymal stromal cells increases proliferation and maintenance of haematopoietic progenitor cells

Mesenchymal stromal cells (MSC) have been suggested to provide a suitable cellular environment for in vitro expansion of haematopoietic stem and progenitor cells (HPC) from umbilical cord blood. In this study, we have simultaneously analysed the cell division history and immunophenotypic differentiation of HPC by using cell division tracking with carboxyfluorescein diacetate N -succinimidyl ester (CFSE). Co-culture with MSC greatly enhanced proliferation of human HPC, especially of the more primitive CD34⁺CD38⁻ fraction. Without co-culture CD34 and CD133 expressions decreased after several cell divisions, whereas CD38 expression was up-regulated after some cell divisions and then diminished in fast proliferating cells. Co-culture with MSC maintained a primitive immunophenotype (CD34⁺, CD133⁺ and CD38⁻) for more population doublings, whereas up-regulation of differentiation markers (CD13, CD45 and CD56) in HPC was delayed to higher numbers of cell divisions. Especially MSC of early cell passages maintained CD34 expression in HPC over more cell divisions, whereas MSC of higher passages further enhanced their proliferation rate. Inhibition of mitogen-activated protein kinase 1 (MAPK1) impaired proliferation and differentiation of HPC, but not maintenance of long-term culture initiating cells. siRNA knockdown of N-cadherin and VCAM1 in feeder layer cells increased the fraction of slow dividing HPC, whereas knockdown of integrin beta 1 (ITGB1) and CD44 impaired their differentiation. In conclusion, MSC support proliferation as well as self-renewal of HPC with primitive immunophenotype. The use of early passages of MSC and genetic manipulation of proteins involved in HPC–MSC interaction might further enhance cord blood expansion on MSC.     

Mesenchymal stem cells derived from different origins have unique sensitivities to different chemotherapeutic agents

BMSCs (bone‐marrow‐derived mesenchymal stem cells) and ADSCs (adipose tissue‐derived mesenchymal stem cells) are virtually identical in cell surface marker profile and differentiation potential. These cell populations have promising characteristics for clinical application. We have investigated the sensitivity of these cell populations to various chemotherapeutic agents by testing the inhibition of cell proliferation, low molecular DNA bands formation, in situ apoptosis, apoptosis‐related gene expression and cell senescence after treatment. BU (busulfan), methotrexate and doxorubicin treatment led to a marked and dose‐dependent reduction in cell viability compared with 5‐FU (5‐fluorouracil) treatment. Different expression patterns of apoptosis‐related genes were found in the BMSCs and ADSCs following treatment with the agents, but no low molecular mass DNA bands were detected. BMSCs had a higher percentage of apoptotic and senescent cells following treatment with chemotherapeutic agents compared with ADSCs. These findings suggest that these two cell populations respond differently to chemotherapy treatment. ADSCs are more resistant than BMSCs to chemotherapy‐induced senescence and apoptosis, indicating that they might be more advantageous to use in the clinic than BMSCs.     

Tetra- and hexavalent mannosides inhibit the pro-apoptotic, antiproliferative and cell surface clustering effects of concanavalin-A: impact on MT1-MMP functions in marrow-derived mesenchymal stromal cells

Mesenchymal stromal cells (MSC) mobilization and recruitment by experimental vascularizing tumors involves membrane type 1-matrix metalloproteinase (MT1-MMP) functions. Given that the mannose-specific lectin Concanavalin-A (ConA) induces MT1-MMP expression and mimics biological lectins/carbohydrate interactions, we synthesized and tested the potential of 11 mannoside clusters to block ConA activities on MSC. We found that tetra- and hexavalent mannosides reversed ConA-mediated changes in MSC morphology and antagonized ConA-induced caspase-3 activity and proMMP-2 activation. Tetra- and hexavalent mannosides also inhibited ConA- but not the cytoskeleton disrupting agent Cytochalasin-d-induced MT1-MMP cell surface proteolytic processing mechanisms, and effects on cell cycle phase progression. The antiproliferative and pro-apoptotic impact of ConA on the MT1-MMP/glucose-6-phosphate transporter signaling axis was also reversed by these mannosides. In conclusion, we designed and identified glycocluster constructions that efficiently interfered with carbohydrate-binding proteins (lectins) interaction with oligosaccharide moieties of glycoproteins at the cell surface of MSC. These glycoclusters may serve in carbohydrate-based anticancer strategies through their ability to specifically target MT1-MMP pleiotropic functions in cell survival, proliferation, and extracellular matrix degradation.     

胎盘间充质干细胞的体外培养及其生物学特性研究

目的：探讨胎盘间充质干细胞（PMSCs）的体外分离和培养方法,建立稳定的PMSCs体外培养扩增体系。方法：将胎盘组织经胶原酶消化、密度梯度离心、贴壁筛选法分离,获得并培养人PMSCs,观察细胞形态及其超微结构;应用流式细胞术测定细胞周期及CD14、CD29、CD34、CD44、CD45的表达,研究其增殖和生长特性。结果：在体外培养条件下,人PMSCs贴壁生长,为成纤维细胞样,与骨髓间充质干细胞相似;CD14/CD34/CD45阴性,CD29/CD44阳性,核浆比大,细胞周期检测G0/G1期约占95%,具有原始细胞的特征。结论：体外获得的PMSCs形态单一、生长稳定、增殖能力较强,具有与骨髓间充质干细胞相似的细胞形态、表面标志。由于其来源方便、丰富,无伦理学限制,因此可进一步用于细胞治疗的研究。     

Defined serum-free media for in vitro expansion of adipose-derived mesenchymal stem cells

BackgroundThere is a growing interest in mesenchymal stem cells (MSCs) because they are regarded as good candidates for cell therapy. Adipose tissue represents an easily accessible source to derive mesenchymal stem cells (Ad-MSCs) non-invasively in large numbers. The aim of this study was to evaluate a defined serum-free medium for in vitro expansion of MSCs as a prerequisite for their clinical use.MethodsAdipose tissue was isolated from healthy donors. Cells were isolated and expanded for five passages in serum-free medium (Mesencult-XF) and Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (DMEM-FBS). MSC morphology, marker expression, viability, population doubling time and differentiation potential toward osteogenic and adipogenic lineages were evaluated. Bone marrow MSCs were included as controls.ResultsAd-MSCs cultured in Mesencult-XF had shorter population doubling time (33.3 ± 13.7 h) compared with those cultured in DMEM-FBS (54.3 ± 41.0 h, P < 0.05). Ad-MSCs cultured in Mesencult-XF displayed a stable morphology and surface marker expression and a higher differentiation potential in comparison to Ad-MSCs cultured in DMEM-FBS.ConclusionsThe defined serum-free and xeno-free Mesencult-XF media appear to be a good choice for Ad-MSCs, but it is not as good in supporting culture of bone marrow MSCs when the cells are to be used for clinical purposes.     

Differential response to hepatic differentiation stimuli of amniotic epithelial cells isolated from four regions of the amniotic membrane

Human Amniotic Epithelial Cells (hAEC) isolated from term placenta are a promising source for regenerative medicine. However, it has long been debated whether the hAEC population consists of heterogeneous or homogeneous cells. In a previous study, we investigated the characteristics of hAEC isolated from four different regions of the amniotic membrane finding significant heterogeneity. The aim of this study was to evaluate the hepatic differentiation capability of hAEC isolated from these four regions. Human term placentae were collected after caesarean section and hAEC were isolated from four regions of the amniotic membrane (R1-R4, according to their relative distance from the umbilical cord) and treated in hepatic differentiation conditions for 14 days. hAEC-derived hepatocyte-like cells showed marked differences in the expression of hepatic markers: R4 showed higher levels of Albumin and Hepatocyte Nuclear Factor (HNF) 4α whereas R1 expressed higher Cytochrome P450 enzymes, both at the gene and protein level. These preliminary results suggest that hAEC isolated from R1 and R4 of the amniotic membrane are more prone to hepatic differentiation. Therefore, the use of hAEC from a specific region of the amniotic membrane should be taken into consideration as it could have an impact on the outcome of therapeutic applications.