系统性硬化症患者骨髓间充质干细胞的生物学特性

目的:研究系统性硬化症(SSc)患者骨髓间充质干细胞(BM-MSC)的生物学特性.方法:从骨髓中分离培养间充质干细胞(MSC),从形态、表面标记和多向分化潜能等方面进行鉴定.通过淋巴母细胞转化、混合淋巴细胞培养研究SSc患者MSC的免疫调节功能,用RT-PCR方法检测MSC免疫相关因子的表达.结果:从4例SSc患者和3...     

Gastritis Promotes an Activated Bone Marrow-Derived Mesenchymal Stem Cell with a Phenotype Reminiscent of a Cancer-Promoting Cell

Background

Bone marrow-derived mesenchymal stem cells (BM-MSCs) promote gastric cancer in response to gastritis. In culture, BM-MSCs are prone to mutation with continued passage but it is unknown whether a similar process occurs in vivo in response to gastritis.

Aim

The purpose of this study was to identify the role of chronic gastritis in the transformation of BM-MSCs leading to an activated cancer-promoting phenotype.

Methods

Age matched C57BL/6 (BL/6) and gastrin deficient (GKO) mice were used for isolation of stomach, serum and mesenchymal stem cells (MSCs) at 3 and 6 months of age. MSC activation was assessed by growth curve analysis, fluorescence-activated cell sorting and xenograft assays. To allow for the isolation of bone marrow-derived stromal cells and assay in response to chronic gastritis, IRG/Vav-1^Cre mice that expressed both enhanced green fluorescent protein-expressing hematopoietic cells and red fluorescent protein-expressing stromal cells were generated. In a parabiosis experiment, IRG/Vav-1^Cre mice were paired to either an uninfected Vav-1^Cre littermate or a BL/6 mouse inoculated with Helicobacter pylori.

Results

GKO mice displayed severe atrophic gastritis accompanied by elevated gastric tissue and circulating transforming growth factor beta (TGFβ) by 3 months of age. Compared to BM-MSCs isolated from uninflamed BL/6 mice, BM-MSCs isolated from GKO mice displayed an increased proliferative rate and elevated phosphorylated-Smad3 suggesting active TGFβ signaling. In xenograft assays, mice injected with BM-MSCs from 6-month-old GKO animals displayed tumor growth. RFP+ stromal cells were rapidly recruited to the gastric mucosa of H. pylori parabionts and exhibited changes in gene expression.

Conclusions

Gastritis promotes the in vivo activation of BM-MSCs to a phenotype reminiscent of a cancer-promoting cell.     

Biocompatible Properties and Mineralization Potential of Premixed Calcium Silicate-Based Cements and Fast-Set Calcium Silicate-Based Cements on Human Bone Marrow-Derived Mesenchymal Stem Cells

Premixed calcium silicate-based cements (CSCs) and fast-set CSCs were developed for the convenience of retrograde filling during endodontic microsurgery. The aim of this study was to analyze the biocompatible properties and mineralization potential of premixed CSCs, such as Endocem MTA Premixed (EM Premixed) and EndoSequence BC RRM putty (EndoSequence), and fast-set RetroMTA on human bone marrow-derived mesenchymal stem cells (BMSCs) compared to ProRoot MTA. Using CCK-8, a significantly higher proliferation of BMSCs occurred only in the EM Premixed group on days 2 and 4 (p < 0.05). On day 6, the ProRoot MTA group had significantly higher cell proliferation than the control group (p < 0.05). Regardless of the experimental materials, all groups had complete cell migration by day 4. Alizarin Red-S staining and alkaline phosphatase assay demonstrated higher mineralization potential of all CSCs similar to ProRoot MTA (p < 0.05). The EndoSequence group showed more upregulation of SMAD1 and OSX gene expression than the other experimental groups (p < 0.05), and all experimental cements upregulated osteogenic gene expression more than the control group (p < 0.05). Therefore, using premixed CSCs and fast-set CSCs as retrograde filling cements may facilitate satisfactory biological responses and comparable osteogenic potential to ProRoot MTA.     

Tryptophan and Kynurenine Enhances the Stemness and Osteogenic Differentiation of Bone Marrow-Derived Mesenchymal Stromal Cells In Vitro and In Vivo

Aging tissues present a progressive decline in homeostasis and regenerative capacities, which has been associated with degenerative changes in tissue-specific stem cells and stem cell niches. We hypothesized that amino acids could regulate the stem cell phenotype and differentiation ability of human bone marrow-derived mesenchymal stromal cells (hBMSCs). Thus, we performed a screening of 22 standard amino acids and found that D-tryptophan (10 μM) increased the number of cells positive for the early stem cell marker SSEA-4, and the gene expression levels of OCT-4, NANOG, and SOX-2 in hBMSCs. Comparison between D- and L-tryptophan isomers showed that the latter presents a stronger effect in inducing the mRNA levels of Oct-4 and Nanog, and in increasing the osteogenic differentiation of hBMSCs. On the other hand, L-tryptophan suppressed adipogenesis. The migration and colony-forming ability of hBMSCs were also enhanced by L-tryptophan treatment. In vivo experiments delivering L-tryptophan (50 mg/kg/day) by intraperitoneal injections for three weeks confirmed that L-tryptophan significantly increased the percentage of cells positive for SSEA-4, mRNA levels of Nanog and Oct-4, and the migration and colony-forming ability of mouse BMSCs. L-kynurenine, a major metabolite of L-tryptophan, also induced similar effects of L-tryptophan in enhancing stemness and osteogenic differentiation of BMSCs in vitro and in vivo, possibly indicating the involvement of the kynurenine pathway as the downstream signaling of L-tryptophan. Finally, since BMSCs migrate to the wound healing site to promote bone healing, surgical defects of 1 mm in diameter were created in mouse femur to evaluate bone formation after two weeks of L-tryptophan or L-kynurenine injection. Both L-tryptophan and L-kynurenine accelerated bone healing compared to the PBS-injected control group. In summary, L-tryptophan enhanced the stemness and osteoblastic differentiation of BMSCs and may be used as an essential factor to maintain the stem cell properties and accelerate bone healing and/or prevent bone loss.     

骨髓间充质干细胞体外诱导分化为心肌细胞

目的观察5-氮杂胞苷(5-aza)体外诱导分化骨髓间充质干细胞(Mesenchymal Stem Cells,MSCs)成为心肌细胞的作用,并为进一步体内研究而进行细胞标记.方法提取小型猪骨髓20ml,体外分离培养骨髓间充质干细胞,分为对照组和诱导组,其中诱导组在培养第3天加入5-aza(10 μmol/L),分别在培养2周和4周时观察两组细胞光镜、电镜下形态,4周时进行磷钨酸-苏木素染色(PTAH)和肌动蛋白(actin)免疫组化染色.诱导组MSCs加入5-溴脱氧尿嘧啶(BrdU,5μg/ml),分别在作用后第2周和第4周,利用BrdU单克隆抗体免疫组化检测BrdU标记方法.此外,用含有LacZ基因的复制缺陷腺病毒转染诱导组MSCs,利用X-gal染色检测LacZ基因表达的半乳糖苷酶,观察诱导分化后MSCs的体外标记情况.结果体外能够成功地分离骨髓MSCs,5-aza诱导分化2周和4周后的MSCs在光镜下明显成为梭形,形态类似肌细胞.电镜下诱导分化后的MSCs在2周时出现肌丝结构,4周时肌丝明显增多.PTAH染色显示超过70%的细胞具有横纹肌的染色性质,actin免疫组化染色显示超过80%的诱导后MSCs肌动蛋白呈阳性表达.体外BrdU单克隆抗体检测BrdU掺入到MSCs细胞核中,X-gal染色证明LacZ基因能够进入MSCs并表达,两种体外标记方法在标记2周和4周时都为阳性.结论5-aza能够诱导骨髓MSCs成为心肌细胞,BrdU掺入和含有LacZ基因的复制缺陷腺病毒转染能够成功对诱导分化后MSCs进行标记,并且标记持续至少4周.     

骨髓间充质干细胞拮抗氧化型低密度脂蛋白

目的 探讨骨髓间充质干细胞(MSC)对氧化型低密度脂蛋白(ox-LDL)诱导的人脐静脉内皮细胞(HUVEC)凋亡的影响及有关机制.方法 将对数生长期的HUVEC分成三组:对照组单纯培养HUVEC;ox-LDL组的HUVEC给予100 mg/L ox-LDL培养24 h;ox-LDL+ MSC组使用细胞培养池共同培养HUVEC和MSC,并加入100mg/L ox-LDL培养24 h.采用流式细胞术检测HUVEC的凋亡情况,采用ELISA检测细胞培养上清液中肿瘤坏死因子α(TNF-α)和血管内皮生长因子(VEGF)的含量,并通过Real-timePCR检测HUVEC中凋亡相关基因Bcl-2与Bax mRNA的表达情况.结果 在100 mg/L ox-LDL作用24 h后,细胞凋亡率明显上升,细胞培养上清液中TNF-α、VEGF含量明显上升,Bcl-2 mRNA表达下调,而Bax mRNA表达上调.MSC与HUVEC共培养可增加上清液中VEGF含量,减少TNF-α含量,上调Bcl-2 mRNA表达,下调Bax mRNA表达,使内皮细胞凋亡率明显下降.结论 MSC可以拮抗ox-LDL诱导的血管内皮细胞凋亡,可能与其分泌VEGF、抑制炎性反应并上调Bcl-2 mRNA表达及下调BaxmRNA表达有关,为MSC治疗动脉粥样硬化等内皮损伤性疾病进一步提供了理论基础.     

骨髓间充质干细胞移植对兔颈动脉球囊损伤后再狭窄的影响

目的 探讨兔颈动脉行球囊损伤后,骨髓间充质干细胞(BMSC)移植对损伤血管内皮修复和再狭窄的影响.方法 建立兔颈动脉粥样硬化狭窄模型48只,随机分成BMSC移植组24只和对照组24只.体外培养BMSC,携带增强型绿色荧光蛋白(EGFP)的腺病毒转染标记后备用.颈总动脉球囊损伤后,以107个/kg的细胞数经颈外动脉移植到损伤动脉局部,对照组注射等量的PBS液.移植后1周取材行免疫组织化学检测BMSC归巢.移植后2周免疫组织化学染色检测血管内膜血小板-内皮细胞黏附分子(CD31)、α-平滑肌肌动蛋白(SM α-actin)及增殖细胞核抗原(PCNA)的表达;移植后4周行颈总动脉造影检测血管狭窄率,HE染色检测损伤血管新生内膜面积、新生内膜面积/中膜面积的变化.结果 BMSC移植组在术后1周损伤血管的内膜有表达EGFP的BMSC归巢.术后2周BMSC移植组血管内膜有连续性CD31的表达,对照组为阴性;BMSC移植组PCNA的表达较对照组明显降低(23.43％±2.80％比50.49％±3.60％,P＜0.05),而BMSC移植组SM α-actin的表达较对照组明显增加(0.437±0.049比0.197 ±0.032,P＜0.01).术后4周HE结果显示:BMSC移植组血管新生内膜面积(0.103±0.022比0.214±0.024,P＜0.01)、新生内膜/中膜面积(0.771±0.096比1.646±0.223,P＜0.01)均较对照组减轻;颈动脉造影结果显示:BMSC移植组较对照组血管再狭窄率减轻(39.64％±2.30％比63.31％±2.82％,P＜0.05).结论 移植BMSC可促进颈动脉球囊损伤后早期再内皮化和血管平滑肌细胞表型转化,抑制血管新生内膜的增生,减轻了血管再狭窄.     

Treatment of Experimental Injury of Anal Sphincters with Primary Surgical Repair and Injection of Bone Marrow-Derived Mesenchymal Stem Cells

Purpose Sphincter injury is a common cause of anal incontinence. Surgical repair remains the operation of choice; however, the outcome often is poor. We investigated the ability of injected bone marrow-derived mesenchymal stem cells to enhance sphincter healing after injury and primary repair in a preclinical model. Methods Twenty-four inbred Wistar Furth rats were divided into three groups. As a control, Group A underwent sham operation. Group B had sphincterotomy and repair of both anal sphincters plus saline injections. The study group (Group C) underwent sphincterotomy and repair followed by intrasphincteric injections of syngenic bone marrow-derived mesenchymal stem cells. A further group (Group D) of outbred Wistar rats treated with mesenchymal stem cells and immunosuppressive therapy also was evaluated. At 30 days, histologic and morphometric analysis and in vitro contractility testing was performed. Results A significant decrease of muscle tissue was observed at the site of repair after sphincter injury. However, in Groups C and D, histologic examination demonstrated new muscle fibers and morphometric analysis revealed a significantly greater muscle area fraction than in Group B (P < 0.05). Moreover, mesenchymal stem cells injection improved contractility of sphincters strips compared with Group B (P < 0.05). No significant differences were found between Groups C and D. Conclusions In our experimental model, bone marrow-derived mesenchymal stem cells injection improved muscle regeneration and increased contractile function of anal sphincters after injury and repair. Therefore, mesenchymal stem cells may represent an attractive tool for treating anal sphincter lesions in humans. Investigations into the biologic basis of this phenomenon should increase our knowledge on underlying mechanisms involved in sphincter repair. Supported by a grant from the University of Siena (PAR 2005). Address of correspondence: Marco Lorenzi, M.D., Department of Surgery, University of Siena, Viale Bracci, 53100 Siena, Italy.     

犬骨髓间叶干细胞的多向分化潜力

目的观察犬骨髓间叶干细胞的体外多向分化潜力,为损伤组织的干细胞移植修复提供理论基础.方法利用梯度-贴壁筛选法分离、培养与扩增骨髓间叶干细胞.利用化学诱导剂5-氮胞苷(20μmol/L),血管内皮细胞生长因子(VEGF 10ng/ml),成骨细胞诱导剂(地塞米松10nMol/L,抗坏血酸0.05mMol,β甘油磷酸钠10mMol/L)以及成脂肪细胞诱导剂(1-甲基3-异丁基黄嘌呤0.5mmol/L,地塞米松1μmol/L,胰岛素10mg/L,消炎痛100mmol/L),分别定向诱导骨髓间叶干细胞分化为心肌细胞、血管内皮细胞、成骨细胞与脂肪细胞.再应用细胞形态观察、免疫细胞化学染色及电镜对分化细胞进行鉴定.结果利用梯度-贴壁筛选法可以分离培养与扩增骨髓间叶干细胞.5-氮胞苷可诱导干细胞呈现肌管、肌丝、心房颗粒,且肌细胞特异性蛋白α-actinin,Myosin,α-Actin,Troponin Ⅰ免疫染色阳性;VEGF可诱导骨髓间叶干细胞出现管网状、血管样及鹅卵石样结构,vWF免疫染色阳性;成骨细胞诱导剂可诱导分化细胞碱性磷酸酶阳性;成脂肪细胞诱导剂使分化细胞内出现脂肪滴,油红O染色示脂滴为橙红色.结论成年犬骨髓间叶干细胞在体外具备多向分化潜力.在化学或生物诱导剂的作用下,犬骨髓间叶干细胞能够定向分化为心肌细胞、血管内皮细胞、成骨细胞、脂肪细胞等多种间叶组织类型细胞.     

Potential of Pluripotent Stem Cells for Diabetes Therapy

Diabetes mellitus type 1 (T1DM) and type 2 (T2DM) are common diseases. To date, it is widely accepted that all forms of DM lead to the loss of beta cells. Therefore, to avoid the debilitating comorbidities when glycemic control cannot be fully achieved, some would argue that beta cell replacement is the only way to cure the disease. Due to organ donor shortage, other cell sources for beta cell replacement strategies have to be employed. Pluripotent stem cells, including embryonic stem (ES) and induced pluripotent stem (iPS) cells offer a valuable alternative to provide the necessary cells to substitute organ transplants but also to serve as a model to study the onset and progression of the disease, resulting in better treatment regimens. This review will summarize recent progress in the establishment of pluripotent stem cells, their differentiation into the pancreatic lineage with a focus on two-dimensional (2D) and three-dimensional (3D) differentiation settings, the special role of iPS cells in the analysis of genetic predispositions to diabetes, and techniques that help to move current approaches to clinical applications. Particular attention, however, is also given to the long-term challenges that have to be addressed before ES or iPS cell-based therapies will become a broadly accepted treatment option.     

Bone Marrow-Derived Stem Cells for Patients with Liver Cirrhosis: A Systematic Review and Meta-analysis

Background: To date, studies have shown inconsistent results of treatment with bone marrow-derived stem cells (BMDSC) for patients with liver cirrhosis. This study aims to compare the efficacy and safety of BMDSC and standard therapy for liver cirrhosis.Methods: Articles from PubMed, Embase, and the Cochrane library were searched from inception to April 2018. The index included Model for End-stage Liver Disease (MELD), alanine aminotransferase (ALT), albumin, total bilirubin (TBIL), prothrombin time (PT), Child–Pugh score, and all-cause mortality.Results: A total of 9 studies with a total of 424 patients with liver cirrhosis were included in final meta-analysis. BMDSC therapy was associated with lower MELD within 3 months (P = .010), while it had no significant impact on MELD after 6 months (P = .074). There were no differences between BMDSC and standard therapy for ALT within 3 months (P = .336) and after 6 months (P = .379). BMDSC did not affect albumin level within 3 months (P = .196) and after 6 months (P = .840). BMDSC reduced the TBIL level within 3 months (P = .037) and was not associated with the TBIL level after 6 months (P = .914). There were no differences between BMDSC and standard therapy for PT within 3 months (P = .167) and after 6 months (P = .484). The Child–Pugh scores within 3 months (P = .342) and after 6 months (P = .133) were not associated with BMDSC treatment for liver cirrhosis patients. Finally, the BMDSC was not associated with the risk of all-cause mortality, as compared with standard therapy (P = .622).Conclusions: BMDSC treatment for patients with liver cirrhosis could improve short-term MELD and TBIL, but not the risk of mortality, as compared with standard therapy.     

Deficient Proliferation of Bone Marrow-Derived Mesenchymal Stem Cells in Patients with Chronic Hepatitis B Viral Infections and Cirrhosis of the Liver

In this study, we determined whether the proliferation of bone marrow-derived mesenchymal stem cells (MSCs) is impaired in patients with chronic hepatitis B viral infection and cirrhosis of the liver. MSCs from 15 patients with chronic hepatitis B and cirrhosis of the liver (CIR-MSCs) and 11 normal donors (ND-MSCs) were collected and characterized in vitro. CIR-MSCs displayed an intact immunophenotype. The percentage of S-phase nuclei in CIR-MSCs (4.34%), however, was significantly lower than that in ND-MSCs (P < 0.001), indicating impaired proliferation of CIR-MSCs. Growth factor receptor expression (e.g., IGF1, PDGFα, and PDGFβ) on the surface of CIR-MSCs decreased compared to that on ND-MSCs (P < 0.03). We found no evidence that CIR-MSCs were infected with the hepatitis B virus (HBV). Deficient proliferation of CIR-MSCs may result from the decreased expression of growth factor receptors and unbalanced production of cytokines in patients with HBV infection. Our results indicate that autologous MSCs of patients with chronic hepatitis B and cirrhosis of the liver may not be suitable for therapeutic purposes.     

Bone Marrow-Derived Stem Cells: a Mixed Blessing in the Multifaceted World of Diabetic Complications

Diabetes is one of the main economic burdens in health care, which threatens to worsen dramatically if prevalence forecasts are correct. What makes diabetes harmful is the multi-organ distribution of its microvascular and macrovascular complications. Regenerative medicine with cellular therapy could be the dam against life-threatening or life-altering complications. Bone marrow-derived stem cells are putative candidates to achieve this goal. Unfortunately, the bone marrow itself is affected by diabetes, as it can develop a microangiopathy and neuropathy similar to other body tissues. Neuropathy leads to impaired stem cell mobilization from marrow, the so-called mobilopathy. Here, we review the role of bone marrow-derived stem cells in diabetes: how they are affected by compromised bone marrow integrity, how they contribute to other diabetic complications, and how they can be used as a treatment for these. Eventually, we suggest new tactics to optimize stem cell therapy.     

Bone Marrow Mesenchymal Stem Cells in Myelodysplastic Syndromes: Cytogenetic Characterization

Aim: This study compared genetic aberrations in hematopoietic cells (HCs) and mesenchymal stem cells of myelodysplastic syndrome (MDS-MSCs) patients. Methods: We obtained chromosomes with aberrations from 22 patients with MDS and chromosomes from 7 healthy individuals. Chromosomal aberrations in both HCs and MSCs were identified using G-banding. We then performed DNA content analysis of the HCs and MSCs. Results: Cytogenetic aberrations were detected in HCs from 13 of the 22 MDS patients (59%). Chromosomal aberrations in MSCs were detected in 15 of the 22 MDS patients (68%). No chromosomal abnormalities were identified in MSCs of the 7 healthy volunteers. We demonstrate herein that MSCs have distinct genetic abnormalities compared to HCs from the same individual. We observed a random loss of chromosomal material in significant proportions of MSCs. A high proportion of random loss may be a marker of chromosomal instability of MDS-MSCs. However, two case results showed that HCs and MSCs have different altered structural changes. Conclusion: Our results suggest enhanced genetic susceptibility of these cells in MDS patients. Our data indicates that the genetic alterations in MSCs may constitute a particular biological mechanism of MDS pathogenesis.     

Mesenchymal Stem Cells in Combination with Scaffolds for Bone Tissue Engineering

This article reviews past and current strategies of the use of bone graft substitutes along with the future biologic alternatives that can enhance the functional capabilities of those grafts. Many of these bone graft substitute alternatives include ceramic-based, allograft-based, factor-based and polymer-based whereas others are cell-based. The ways of achieving the goal of tissue engineering using stem cells and their lineage to regenerate tissue have been detailed with regard to both the generation of sufficient vascular invasion of the tissue to improve oxygen and nutrient supply, and the development of innovative physical/chemical stimuli to induce bone formation with the proper biomaterial to carry the cells. It is imperative to integrate basic polymer science with molecular biology and stem cell biology, in the design of new materials that perform very sophisticated signaling needed for integration and function.