Little evidence of transdifferentiation of bone marrow-derived cells into pancreatic beta cells

Aims/hypothesis Bone marrow cells contain at least two distinct types of stem cells which are haematopoietic stem cells and mesenchymal stem cells. Both cells have the ability to differentiate into a variety of cell types derived from all three germ layers. Thus, bone marrow stem cells could possibly be used to generate new pancreatic beta cells for the treatment of diabetes. In this study, we investigated the feasibility of bone marrow-derived cells to differentiate into beta cells in pancreas.Methods Using green fluorescent protein transgenic mice as donors, the distribution of haematogenous cells in the pancreas was studied after bone marrow transplantation.Results In the pancreas of green fluorescent protein chimeric mice, green fluorescent protein-positive cells were found in the islets, but none of these cells expressed insulin. Previous data has suggested that tissue injury can recruit haematopoietic stem cells or their progeny to a non-haematopietic cell fate. Therefore, low-dose streptozotocin (30 or 50 mg/kg on five consecutive days) was injected into the mice 5 weeks after bone marrow transplantation, but no green fluorescent protein-positive cells expressing insulin were seen in the islets or around the ducts of the pancreas.Conclusions/interpretation Our data suggests that bone marrow-derived cells are a distinct cell population from islet cells and that transdifferentiation from bone marrow-derived cells to pancreatic beta cells is rarely observed.Abbreviations STZ streptozotocin - EGFP enhanced green fluorescent protein - GP guinea-pig - vWF von Willebrand Factor - BrdU bromodeoxyuridine - GFP green fluorescent protein - IPGTT Intraperitoneal glucose tolerance test     

Hematopoietic Progenitor Cells Residing in Muscle Engraft into Bone Marrow following Transplantation

Hematopoietic and mesenchymal stem cells can potentially be the same cell type or adhere simultaneously in both bone marrow (BM) and muscle. In this study, we asked whether murine BM-derived cells could be tracked in muscle tissue after BM transplantation and whether muscle-derived cells have hematopoietic potential. To answer the first question, we transplanted BM from male BALB/c mice into irradiated female recipients and analyzed for engraftment. We used quantitative polymerase chain reaction (PCR) and fluorescent in situ hybridization techniques for Y chromosome-specific gene probes. A high number of BM-derived cells were located in both the intravascular and extravascular spaces in muscle tissue after BM transplantation. To answer the second question, we analyzed colony-forming potential in vitro with soft-agar assays and the competitive engraftment potential in vivo of muscle-derived cells. Engraftment levels of male cell populations were tested by quantitative PCR. The long-term engraftment potential of muscle-derived cells was low compared with that of BM. We conclude that there is intensive cellular trafficking between BM and muscle tissue. The engraftment potential of muscle-derived stem cells into BM is low and corresponds to the low amounts of hematopoietic colony-forming cells found in muscle tissue.     

骨髓间充质干细胞与心肌修复

骨髓间充质干细胞 (MSC)具有多向分化潜能 ,在一定的诱导条件下可以分化为多种细胞。 5 氮胞苷可以诱导离体MSC出现心肌表型。MSC移植研究表明 ,在心肌组织微环境中 ,MSC可向心肌分化并与宿主心肌功能整合 ,从而修复损伤坏死心肌、改善心脏功能     

Stem Cell Plasticity in the Hematopoietic System

Bone marrow (BM) contains hematopoietic stem cells, which differentiate into all mature blood cells, and marrow stromal cells that provide the microenvironment for hematopoietic stem/progenitor cells along with the capability to differentiate into mature cells of multiple mesenchymal tissues including fat, bone, and cartilage. Recent studies indicate that adult BM also contains cells that can differentiate into nonhematopoietic cells of ectodermal, mesodermal, and endodermal tissues other than hematopoietic tissues, including liver, pancreas, kidney, lung, skin, gastrointestinal tract, heart, skeletal muscles, and neural tissues. Studies reporting the multipotentiality of BM cells have become a focus of interest because they suggest that clinical applications could be at hand using easily obtainable cells in the treatment of tissue damage or degenerative diseases. Presently, however, definitive evidence explaining the mechanism of this multipotentiality of BM stem cells is lacking. In this review, we summarize recent progress and controversies in investigation of the multipotentiality of adult BM-derived stem cells to differentiate into nonhematopoietic tissues.     

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.     

Mesenchymal Stem Cells Derived from Bone Marrow of Diabetic Patients Portrait Unique Markers Influenced by the Diabetic Microenvironment

Cellular microenvironment is known to play a critical role in the maintenance of human bone marrow-derived mesenchymal stem cells (BM-MSCs). It was uncertain whether BM-MSCs obtained from a ''diabetic milieu'' (dBM-MSCs) offer the same regenerative potential as those obtained from healthy (non-diabetic) individuals (hBM-MSCs). To investigate the effect of diabetic microenvironment on human BM-MSCs, we isolated and characterized these cells from diabetic patients (dBM-MSCs). We found that dBM-MSCs expressed mesenchymal markers such as vimentin, smooth muscle actin, nestin, fibronectin, CD29, CD44, CD73, CD90, and CD105. These cells also exhibited multilineage differentiation potential, as evident from the generation of adipocytes, osteocytes, and chondrocytes when exposed to lineage specific differentiation media. Although the cells were similar to hBM-MSCs, 6% (3/54) of dBM-MSCs expressed proinsulin/C-peptide. Emanating from the diabetic microenvironmental milieu, we analyzed whether in vitro reprogramming could afford the maturation of the islet-like clusters (ICAs) derived from dBM-MSCs. Upon mimicking the diabetic hyperglycemic niche and the supplementation of fetal pancreatic extract, to differentiate dBM-MSCs into pancreatic lineage in vitro, we observed rapid differentiation and maturation of dBM-MSCs into islet-like cell aggregates. Thus, our study demonstrated that diabetic hyperglycemic microenvironmental milieu plays a major role in inducing the differentiation of human BM-MSCs in vivo and in vitro.     

骨髓间充质干细胞与糖尿病及其并发症的治疗

传统的药物治疗不能阻止糖尿病患者胰岛β细胞的进行性衰竭.骨髓间充质干细胞是一个缺乏造血干细胞的细胞群,具有自我更新能力以及多向分化潜能,可定向诱导分化为胰岛素分泌细胞,分泌胰岛素,从而可能用于糖尿病及其并发症的治疗.胰-十二指肠同源盒因子-1可提高其分化效率.本文主要对骨髓间充质干细胞治疗糖尿病及其并发症作一综述.     

骨髓基质干细胞向心肌细胞分化的研究进展

骨髓基质干细胞不仅有一定的自我更新能力,可以分化为骨、软骨和脂肪等多种类型的基质细胞,在一定外界环境条件下还能实现跨系统分化,分化成心肌细胞,是目前认为较有希望成为心肌梗死替代治疗的干细胞。现就骨髓基质干细胞的生物学特性、向心肌细胞分化的诱导因素及临床应用进展进行综述。     

Haematopoietic stem cells participate in muscle regeneration

It has previously been shown that bone marrow cells contribute to skeletal muscle regeneration, but the nature of marrow cell(s) involved in this process is unknown. We used an immunocompetent and an immunocompromised model of bone marrow transplantation to characterize the type of marrow cells participating regenerating skeletal muscle fibres. Animals were transplanted with different populations of marrow cells from Green Fluorescent Protein (GFP) transgenic mice and the presence of GFP(+) muscle fibres were evaluated in the cardiotoxin-injured tibialis anterior muscles. GFP(+) muscle fibres were found mostly in animals that received either CD45(-), lineage(-), c-Kit(+), Sca-1(+) or Flk-2(+) populations of marrow cells, suggesting that haematopoietic stem cells (HSC) rather than mesenchymal cells or more differentiated haematopoietic cells are responsible for the formation of GFP(+) muscle fibres. Mac-1 positive population of marrow cells was also associated with the emergence of GFP(+) skeletal muscle fibres. However, most of this activity was limited to either Mac-1(+) Sca(+) or Mac-1(+)c-Kit(+) cells with long-term haematopoietic repopulation capabilities, indicating a stem cell phenotype for these cells. Experiments in the immunocompromised transplant model showed that participation of HSC in the skeletal muscle fibre formation could occur without haematopoietic chimerism.     

自体骨髓间叶干细胞诱导分化移植重建窦房结起搏功能的实验研究

目的:探索应用骨髓干细胞治疗病态窦房结综合征的生物介入方法.方法:选取犬6只,随机分为实验组和对照组,每组3只.抽取犬自体骨髓,分离培养扩增骨髓间叶干细胞,并在体外应用5-氮胞苷进行诱导分化.应用射频技术损伤犬窦房结,建立动物病态窦房结综合征模型.将溴脱氧尿嘧啶核苷(BrdU)标记的且诱导分化的骨髓间叶干细胞自体移植到窦房结区,应用心电图、组织病理、免疫组化等技术观察干细胞移植治疗效果.结果:在动物病态窦房结综合征模型,自体移植骨髓干细胞后,心电图示窦房结功能明显改善;病理与免疫组化示移植的骨髓干细胞在窦房结区分化为拟窦房结细胞与血管内皮细胞,并与宿主细胞建立缝隙连接.结论:自体移植诱导分化的骨髓间叶干细胞可改善窦房结自律起搏功能.     

Induction of Umbilical Cord Blood Mesenchymal Stem Cells into Neuron-Like Cells In Vitro

Mesenchymal stem cells (MSCs) in human umbilical cord blood are multipotent stem cells that differ from hematopoietic stem cells. They can differentiate in vitro into mesenchymal cells such as osteoblasts and adipocytes. However, differentiation into nonmesenchymal cells has not been demonstrated. Here, we report the isolation, purification, expansion, and differentiation of human umbilical cord blood MSCs into neurocytes in vitro. Cord blood samples were allowed to drain from the end of the cord into glass bottles with 20 U/mL preservative-free heparin. MSCs were isolated from human umbilical cord blood, purified, and expanded in Mesencult medium. Surface antigens of MSCs were analyzed by fluorescence-activated cell sorting (FACS). MSC passages 2,5, and 8 were induced to differentiate into neuron-like cells. Neurofilament (NF) and neuron-specific enolase (NSE) were detected by immunohistochemistry staining. Special Nissl bodies were observed by histochemical analysis. The results showed that 6.6 x 10(5) primary MSCs were expanded for 10 passages to obtain 9.9 x 10(8) MSCs, an increase of approximately 1.5 x 10(3)-fold. FACS results showed that the MSCs did not express antigens CD34, CD11a, and CD11b and expressed CD29 and CD71, an expression pattern identical to that of human bone marrow-derived MSCs. Induction results indicated that approximately 70% of the cells exhibited a typical neuron-like phenotype. Immunohistochemistry staining suggested that induced MSCs of different passages expressed NF and NSE. Special Nissl bodies were obvious in the neuron-like cells. These results suggest that MSCs in human umbilical cord blood are capable of differentiating into neuron-like cells in vitro.     

骨髓干细胞动员治疗急性心肌梗死研究进展

研究发现骨髓干细胞可以分化为心肌细胞、血管内皮细胞以及平滑肌细胞 ,从而为骨髓干细胞治疗心肌梗死提供了基本的理论依据。此外 ,一些研究表明心肌梗死早期伴随有自发性骨髓干细胞的梗死区定向迁移 ,而由于目前已知的多种骨髓干细胞移植方案具有一定的复杂性和潜在危险性 ,动员骨髓干细胞治疗急性心肌梗死成为目前研究的前沿问题。     

骨髓间充质干细胞向神经元样细胞分化中褪黑素受体和多效蛋白的表达

目的体外诱导成人骨髓间充质干细胞（MSCs）向神经元样细胞分化,并探讨分化过程中多效蛋白（PTN）和褪黑素（Mel）受体亚型MT1、MT2 mRNA的表达,以探索MSCs向神经元样细胞分化的特性和机制。方法密度梯度离心加贴壁培养法分离成人MSCs,原代和传代培养。取第6代MSCs设对照和实验组进行诱导,诱导后30min至3d,观察细胞形态并计数。免疫细胞化学法和RT—PCR法测定分化后细胞神经细胞特异性表面标志神经元特异性烯醇化酶（NSE）、微管相关蛋白-2（MAP-2）、神经胶质纤维酸性蛋白（GFAP）和诱导前、诱导后12hPTN和MT1、MT2 mRNA的表达。结果接种24h后MSCs开始贴壁,呈圆形或椭圆形。3d后可见梭状细胞呈集落状生长,10d～14d融合。第5代～第6代时呈现较均一的成纤维细胞样形态。诱导后胞体向胞核收缩;出现双极及多极细胞。12h变形细胞增多,细长突起相互连接。24h后变形细胞增多不明显。诱导后12h大部分细胞表达NSE、MAP-2,未检测到GFAP的表达。实验组诱导后12h细胞有PTN和MT1 mRNA的表达。结论PTN和Me1信号传导可能参与调控了MSCs向神经元样细胞的分化。     

骨髓基质干细胞对H_2O_2诱导的肝细胞损伤保护作用的研究

背景:骨髓基质干细胞是一种多能干细胞,不但可分化为肝细胞,并且可通过旁分泌的方式分泌细胞因子,从而促进肝再生。目的:探讨骨髓基质干细胞对H_2O_2诱导的肝细胞损伤的保护作用。方法:以500μmol/L H_2O_2诱导人肝细胞株THLE-3损伤模型,24h后与骨髓基质干细胞共培养。倒置相差显微镜下观察细胞形态改变,检测细胞毒性、caspase-3/7活性以及YO-PRO-1阳性细胞率。结果:与骨髓基质干细胞共培养后,H_2O_2损伤的THLE-3细胞形态明显改善,细胞毒性显著减少(P0.05),caspase-3/7活性显著下降(P0.05),YO-PRO-1阳性细胞率显著降低(P0.05)。结论:骨髓基质干细胞通过降低细胞毒性、caspase-3/7活性和细胞凋亡而对H_2O_2诱导的THLE-3细胞损伤起保护作用。     

骨髓间充质干细胞移植加重大鼠主动脉血管成形术后再狭窄程度

目的探讨骨髓间充质干细胞（MSCs）移植对大鼠血管成形术后再狭窄的影响。方法随机将大鼠分为正常对照、损伤和损伤加MSCs移植组,用经皮冠状动脉腔内成形术（PTCA）导管扩张大鼠胸腹主动脉创建动脉损伤模型。贴壁法培养大鼠MSCs,经4,6-联脒-2-苯基吲哚（DAPI）荧光标记后,以2×10^6数量经导管注入移植组大鼠主动脉内。术后第1、2、6周取材寻找DAPI标记的MSCs,行组织形态学及血小板衍生的生长因子受体（PDGFR）家族干细胞因子的受体（c—kit）,增殖细胞核抗原（PCNA）,平滑肌肌动蛋白（αSMA）免疫组化分析。结果移植术后1～2周,可见DAPI标记MSCs归巢到损伤内膜表面或中膜近内膜处,并表达d—SMA。移植组和损伤组新生内膜表面均表达少量c-kit,两组之间无明显差别。正常对照组血管中膜α-SMA均匀强表达,PCNA表达微弱,无新生内膜。移植组新生内膜中PCNA和α—SMA表达均强于损伤组。术后6周,移植组新生内膜／中膜面积百分比和管腔面积狭窄率均较损伤组明显增加,差异具有统计学意义。结论经主动脉移植的骨髓MSCs可归巢到严重受损的主动脉内膜上,并分化为平滑肌样细胞,参与新生内膜的形成,加重大鼠血管成形术后再狭窄程度。     

A Novel Approach to Transplanting Bone Marrow Stem Cells to Repair Human Myocardial Infarction: Delivery via a Noninfarct‐relative Artery

Bone marrow stem cells are able to repair infarcted human myocardium following intracoronary transplantation via the infarct‐relative artery. However, traditional reperfusion strategies fail to open the artery in some patients, making effective delivery impossible. Our previous study demonstrated a safe and efficient approach to delivering bone marrow stem cells via a noninfarcted artery in an animal myocardial infarction model. The objective of the present study was to evaluate the safety and feasibility of autologous bone marrow mesenchymal stem cell transplantation via such an approach in patients with acute myocardial infarction (AMI). Sixteen patients with anterior AMI who had successfully undergone percutaneous coronary intervention (PCI) were enrolled in this pilot, randomized study. Three weeks after PCI, cultured bone marrow mesenchymal stem cells were injected into the myocardium via either the infarct‐relative artery (left anterior descending branch artery, LAD) or a noninfarct‐relative artery (right coronary artery, RCA). The safety and feasibility of the cell infusion were evaluated during the procedure and during 6 months of follow‐up. In addition, 2D echocardiography, technetium‐99m methoxyisobutylisonitrile (99mTc‐MIBI) and 18F‐deoxyglucose single photon emission computed tomography were employed to examine cardiac function, myocardial perfusion, and viable cardiomyocytes, respectively, at day 4 after PCI and 6 months after the cell infusion. There were no arrhythmia and any other side‐effects, including infections, allergic reactions or adverse clinical events, during, immediately after, or 6 months after cell transplantation. Cardiac function and myocardial perfusion had improved 6 months after PCI/bone marrow stem cells transplantation. Viable cardiomyocytes metabolism was detected in the infarcted areas in both groups after the cell infusion, as demonstrated by 18F‐deoxyglucose. Intracoronary infusion of autologous bone marrow mesenchymal stem cells via a noninfarct‐relative artery appears safe and feasible in the treatment of patients with AMI.     

Diverse contribution of bone marrow cells to neointimal hyperplasia after mechanical vascular injuries

We and others have suggested that bone marrow-derived progenitor cells may contribute to the pathogenesis of vascular diseases. On the other hand, it was reported that bone marrow cells do not participate substantially in vascular remodeling in other experimental systems. In this study, three distinct types of mechanical vascular injuries were induced in the same mouse whose bone marrow had been reconstituted with that of GFP or LacZ mice. All injuries are known to cause smooth muscle cell (SMC) hyperplasia. At 4 weeks after wire-mediated endovascular injury, a significant number of the neointimal and medial cells derived from bone marrow. In contrast, marker-positive cells were seldom detected in the lesion induced by perivascular cuff replacement. There were only a few bone marrow-derived cells in the neointima after ligation of the common carotid artery. These results indicate that the origin of intimal cells is diverse and that contribution of bone marrow-derived cells to neointimal hyperplasia depends on the type of model.     

The Hematopoietic Microenvironment: The Osteogenic Compartment of Bone Marrow: Cell Biology and Clinical Application

The marrow stromal cells of the bone cavities are of mesenchymal origin. These cells are maintaining bone remodeling and control the differentiation of hemopoietic cells during the life span. The function of the marrow stroma is based on cells that differentiate into various subtypes that are derived from a common stem cell. Plating the bone marrow at low density, they form colonies with a fibroblast-like appearance (CFU-F). The fibroblastic colonies may differentiate to distinct cell types as fibroblasts, endothelial, osteogenic and adipocytes. Understanding of the control mechanism of these cells' differentiation is very important. We used an in vitro system from established primary cultures and continuos cell lines, MBA cells from mouse bone marrow. The MBA cells were used to study the stromal cells subtypes. Herein, we summarized the knowledge on the osteogenic cells' differentiation using the marrow-derived stromal osteogenic cells, MBA-15 and clonal lines. The characterization of cells was based on morphology, rate of cell growth, gene expression and biochemical profile. A deeper understanding of the osteogenic cells differentiation will lead to better use of the marrow stroma cells in clinical application and tissue engineering to ensure healing of bone defects or skeletal genetic diseases.     

Differentiation of Rabbit Bone Marrow Mesenchymal Stem Cells to Myogenic Cells and Expression of VEGF After Gene Transfection

INTRODUCTIONOriginally identified as a source of osteoprogenitorcells, mesenchymal stem cells (MSCs) coulddifferentiate to adipocytes, chondrocytes, osteoblastsand myoblasts in vitro and undergo differentiation invivo[1], making these stem cells promising candidates formesodermal defect repair and disease management.MSCs have been proposed as an alternative source forthe regeneration of myocardium. There were severalclinical studies of MSCs' effects on ischemic heartdisease[2]. However…