中胚层细胞谱系研究

深入认识中胚层细胞谱系产生和发育分化过程有助于体外获得大量可用于移植的、有功能的中胚层来源成体干细胞,为基础科学和再生医学治疗提供理论指导。中国科学院战略性科技先导专项"干细胞与再生医学研究"项目内的中胚层研究,以肌肉和血液两个中胚层来源细胞谱系决定为切入点,探讨中胚层细胞谱系在发育分化过程中的重要功能和调控机制,建立成体干细胞体外扩增培养体系,为中胚层来源干细胞的自我更新、迁移增殖和定向分化研究提供重要的理论基础,并为中胚层系统疾病提供治疗靶点和细胞来源。现将对先导项目启动五年来开展的研究内容及其取得的研究成果进行介绍。     

前列腺类器官技术(POT)——前列腺干细胞和癌症研究中的“新金桶”（英文）

类器官是由单个干细胞、祖细胞或不同谱系的细胞簇在三维培养条件下自发形成类似体内组织结构的细胞簇。近年来,类器官技术为干细胞和癌症领域带来了革命性的变化。在这篇综述中,我们简要地概述了前列腺类器官技术(prostate organoid technology, POT)在前列腺研究中的进展。此外,我们也总结了POT在探究前列腺干细胞和癌症生物学以及探索前列腺癌治疗策略方面的潜在应用。最后,我们就当前POT在应用过程中的技术短板进行了讨论,并为第二代POT在研究前列腺癌细胞行为和患者对药物反应中的用途提供了全新的视角。     

细胞谱系重编程研究进展

重编程研究是近年来干细胞与再生医学领域中里程碑式的研究突破,打破了人胚胎干细胞研究带来的伦理桎梏,为再生医学临床应用带来了一片新天地.但诱导性多能干细胞(induced pluripotent stem cells,iPS)制作过程繁琐,重编程效率偏低,且仍需如同胚胎干细胞一样,经过步步诱导分化而获得目的细胞,最关键的是,目的细胞的安全性问题仍亟待解决.谱系重编程,在广义的角度上来说,至少从1987年即已展开,20世纪末引发成体干细胞研究热潮的"可塑性"研究也可归为谱系重编程研究,在iPS重编程研究的推动下,谱系重编程技术正在蓬勃发展,并被再生医学研究者给予了厚望,尤其是在可预见的将来,其在个体化治疗上具有的优势将不可比拟.     

颌突外胚间充质干细胞(EMSCs)的体外分离及鉴定

外胚间充质(ectomesenchyme)是一种胚胎发育早期颅面部出现的多能性结构(multipotentstructure),大多数颅面部结构和组织均由其衍生而来,这提示外胚间充质中存在一种干细胞,即外胚间充质干细胞(ectomesenchymalstemcells,EMSCs)。为了分离和鉴定EMSCs,对E125的SD大鼠颌突组织细胞进行了流式细胞学分析,发现其中的外胚间充质细胞表达多种神经谱系和中胚层谱系的标志,包括p75、CD57和nestin等。根据此特点,采用磁细胞分离技术对p75+的颌突外胚间充质细胞进行了分离和克隆培养。克隆分析表明,单个p75+细胞经过10～14d培养,可以形成由两种或两种以上细胞组成的多潜能性克隆(multipotentclone),提示该群外胚间充质细胞具有多潜能性。同时,亚克隆分析表明,多潜能性子克隆中的单个p75+细胞具有再次形成多潜能性克隆的能力,说明这些细胞在体外具有自我更新的能力。这些结果提示,p75+细胞同时具有多潜能性和自我更新能力,因此是外胚间充质干细胞。该干细胞的分离对于口腔颅面部的起源和发育研究无疑具有重要意义。此外,该干细胞的高度可塑性也预示它可以作为一种新的种子细胞,为组织工程皮肤、肌肉、软骨的研究提供新思路。     

hESCs/hiPSCs体外诱导产生红细胞的研究进展及其临床应用的展望

人类胚胎干细胞和多功能诱导性干细胞的诞生,标志着干细胞研究已经跨入了全新的应用时代。干细胞研究领域的一个重要方向是特定谱系成熟细胞的定向诱导分化。在诸多的血细胞中,成熟红细胞因为无核而携带着最小量的遗传物质,可能作为最早的干细胞治疗产品而应用于输血替代治疗。最近,干细胞向造血细胞（包括红细胞）的研究正方兴未艾。但由于方法学上的偏差,诱导产生的红细胞的成熟度各有所不同。该文在结合了作者实验室的工作经验的基础上,对目前人类多潜能干细胞向红细胞特定诱导分化的方法做了综合的描述,并提出了该研究领域亟需解决的重大科学问题。     

肠道干细胞和潘氏细胞在肠道稳态和疾病中的作用

肠道是最复杂的器官之一,负责营养的吸收和消化。肠道具有多层结构保护整个肠道免受病原体的侵害。肠道上皮是由单层柱状上皮细胞组成,是抵抗病原体的第一道屏障。因此,肠上皮必须保持完整性以保护肠免受感染和毒性剂的侵害。上皮细胞分为两个谱系(吸收型与分泌型),并且每隔3~4天脱落至肠腔中。细胞的快速更替是由于肠道干细胞的存在,肠道干细胞排列在隐窝底部终极分化的潘氏细胞之间并沿隐窝绒毛轴分化成不同的上皮细胞。一旦肠道干细胞受到损伤,潘氏细胞将通过提供WNT配体和Notch刺激来补充肠道干细胞。因此,潘氏细胞充当辅助细胞以维持干细胞微环境,即生态位。该综述探讨了干细胞和潘氏细胞之间的相互作用,进一步探讨了维持肠道稳态的信号通路。     

终末期肝病细胞治疗供体细胞来源的研究进展

许多因素所致的肝脏疾病都有可能发展为终末期肝病,而终末期肝病目前唯一有效的治疗方法就是原位肝移植。然而,由于受供体肝脏短缺等因素的限制,细胞治疗方法一直被临床上所期待。近年来,基于谱系重编程的诱导型肝干细胞和诱导型肝实质细胞的技术体系的出现,为解决肝脏疾病细胞治疗中细胞来源匮乏的问题提供了新的思路,也加速了肝脏疾病细胞治疗研究向临床转化的进程。该文介绍了肝脏疾病细胞治疗研究中供体细胞来源的现状,并重点介绍了谱系重编程获取肝系细胞的研究进展。     

心肌干细胞与心脏再生的研究进展及展望

以心肌梗死为代表的心血管疾病是目前世界上的主要致死疾病,它是由于心脏的主要功能细胞——心肌细胞的大量死亡造成的,因此如何再生心肌细胞从而促进心脏再生是心脏领域急需解决的一个重要科学问题.寻找能够分化形成心肌细胞的干细胞——心肌干细胞来再生心脏成为近20年来研究的热点问题.近年来,多种类型的干细胞,如c-Kit~+, Sca1~+, Abcg2~+, Bmi1~+等干细胞被报道是内源性的成体心肌干细胞,其中以"c-Kit~+心肌干细胞"的相关研究最为广泛,众多基础研究和临床试验纷纷展开.然而近年来,随着体内遗传谱系示踪技术的发展,越来越多的实验结果表明,成年哺乳动物心脏缺乏具有生物学意义的内源性心肌干细胞.本文主要介绍了成体心肌干细胞的相关研究,并展望了今后心脏再生策略的发展方向.     

成体干细胞在泌尿系统疾病修复与重建中的应用

干细胞是目前生命科学研究的热点方向。干细胞具有自我更新及定向分化的潜在能力。近年来,干细胞移植治疗在治疗压力性尿失禁和膀胱损伤方面已成为研究重点,不同来源的干细胞在治疗膀胱损伤已取得瞩目的研究成果。干细胞对阴茎勃起神经和海绵体血管内皮细胞起着修复保护作用。干细胞具有向多种谱系细胞转化的能力来治疗压力性尿失禁。干细胞移植为泌尿系统的神经肌肉疾病的修复重建提供了一条新途径,使认为不可修复的的神经肌肉疾病的结构修复和组织重建成为可能。干细胞包括脂肪干细胞(adipose-derived stem cells,ADSCs)、骨髓间充质干细胞(bone narrow mesenchymal stem,BMSCs)和肌源性干细胞(muscle-derived stem cell,MDSCs)等。组织工程学是一类交叉学科,主要包括综合细胞培养、材料构建和细胞种植等。组织工程技术为泌尿外科临床医师提供了一条修复乃至重建受损脏器的新思路。本文就利用干细胞作为种子细胞,对膀胱缺损、压力性尿失禁、勃起功能障碍泌尿系疾病的组织工程修复进行综述。     

机械力及力学信号转导影响干细胞命运的研究进展

干细胞作为一种未分化的祖细胞,目前已被广泛应用于开展组织损伤修复、再生以及干细胞特异谱系分化的研究.大量研究表明,干细胞所处的微环境对调控干细胞的生长和分化具有重要作用,多种溶液介质、细胞外基质和信号通路等参与了干细胞命运的调控.尽管已有大量研究证明,溶液介质(如激素和生长因子)在干细胞的生长和分化中发挥重要作用,但近年来越来越多的研究表明,机械力及力学信号转导同样在干细胞自我更新、分化、衰老和凋亡等细胞生理过程中起到重要的作用.本文将对机械应力响应的细胞基础、生物力学及力学信号调控干细胞自我更新和分化,以及生物力学调控干细胞命运可能的作用机制几个方面加以综述.     

Erratum to: Mechanistic insight of platelet apoptosis leading to non-surgical bleeding among heart failure patients supported by continuous-flow left ventricular assist devices

Cardiosphere-derived cells (CDCs) and bone marrow mesenchymal stem cells (MSCs) are popularly used in stem cell therapy for myocardial regeneration. The cell type that survives and maintains stem cell characteristics in the adverse microenvironment following ischemia–reperfusion injury is presumed to be ideal for transplantation. The study was therefore aimed at identifying the cell type with relatively greater resistance to ischemia–reperfusion injury. CDCs were isolated from the right atrial appendage and MSCs from bone marrow of patients who underwent coronary artery bypass graft surgery. Ischemia–reperfusion injury was simulated in vitro by subjecting the cells to hypoxia (0.5% O₂) followed by reintroduction of oxygen (HR injury). Greater resistance of CDCs to HR injury was apparent from the decreased expression of senescence markers and lower proportion of apoptotic cells (one-sixth of that in MSCs). HR injury retarded cell cycle progression in MSCs. Consequent to HR injury, cell migration and secretion of stromal-derived growth factor were stimulated, significantly in CDCs. The differentiation to myocyte lineage and angiogenesis assessed by tube formation ability was better for CDCs. Release of vascular endothelial growth factor was relatively more in CDCs and was further stimulated by HR injury. Differentiation to osteogenic and angiogenic lineage was stimulated by HR injury in MSCs. Compared to MSCs, CDCs appear to be the cell of choice for promoting myocardial regeneration by virtue of its survival capacity in the event of ischemic insult along with higher proliferation rate, migration efficiency, release of growth factors with paracrine effects and differentiation to cardiac lineage.     

Autologous stem cell transplantation for myocardial repair

Current therapies for heart failure due to transmural left ventricular (LV) infarction are limited. We have developed a novel patch method for delivering autologous bone marrow stem cells to sites of myocardial infarction for the purpose of improving LV function and preventing LV aneurysm formation. The patch consisted of a fibrin matrix seeded with autologous porcine mesenchymal stem cells labeled with lacZ. We applied this patch to a swine model of postinfarction LV remodeling. Myocardial infarction was produced by using a 60-min occlusion of the left anterior descending coronary artery distal to the first diagonal branch followed by reperfusion. Results were compared between eight pigs with stem cell patch transplantation, six pigs with the patch but no stem cells (P), and six pigs with left anterior descending coronary artery ligation alone (L). Magnetic resonance imaging data collected 19 +/- 1 days after the myocardial infarction indicated a significant increase of LV systolic wall thickening fraction in the infarct zone of transplanted hearts compared with P or L hearts. Blue X-gal staining was observed in the infarcted area of transplanted hearts. PCR amplification of specimens from the X-gal-positive area revealed the Ad5 RSV-lacZ vector fragment DNA sequence. Light microscopy demonstrated that transplanted cells had differentiated into cells with myocyte-like characteristics and a robust increase of neovascularization as evidenced by von Willebrand factor-positive angioblasts and capillaries in transplanted hearts. Thus this patch-based autologous stem cell procedure may serve as a therapeutic modality for myocardial repair.     

Saphenous vein bypass grafting for coronary atherosclerosis: clinical experience

Saphenous vein bypass grafting is a recent and important procedure in the management of atherosclerotic coronary artery disease. A review of the first 150 patients operated on to July 1971 at the Toronto General Hospital is presented. Many had multiple bypass grafts and some had additional procedures including internal mammary artery implantation, valve replacement and scar tissue resection. There were five operative deaths (3%) and an additional five hospital deaths; the majority were related to myocardial infarction.A clinical review of the results six months to three years after operation indicates marked improvement in over 80% of the survivors. Postoperative hemodynamic studies were performed in many. It is suggested that patients with poor myocardial function presenting in failure may not benefit from the operation.     

Elderly and younger patients selected to undergo coronary angiography.

OBJECTIVE--To establish and compare the characteristics of older (greater than or equal to 70 years) and younger patients with chest pain selected to undergo coronary angiography and by analysis of their subsequent management to assess the value of coronary angiography for older patients with chest pain. DESIGN--Retrospective analysis of clinical case notes and coronary angiography reports. SETTING--Cardiology department with referral population of one million in an Edinburgh hospital. PATIENTS--134 consecutive patients with chest pain aged 70 years or over investigated by coronary angiography between 1978 and 1988; 134 randomly selected patients aged under 70 investigated over the same period. MAIN OUTCOME MEASURES--Clinical and angiographic features at time of angiography and management after angiography. RESULTS--Older patients represented a small, but increasing, proportion of those investigated. Older patients had more severe symptoms at the time of angiography, were taking more antianginal drugs, and had had their symptoms for longer than younger patients. At angiography more older patients had triple vessel coronary disease, left main stem stenosis, or left ventricular impairment. After angiography similar proportions of older and younger patients underwent coronary artery surgery, with more elderly patients requiring urgent operation; although operative mortality was higher for elderly patients, symptomatic benefit was similar to that in younger patients. CONCLUSIONS--Older patients with angina selected to undergo coronary angiography and subsequent coronary surgery have more severe symptoms and underlying cardiac disease. Earlier referral and investigation might yield a population with lower operative risk. Selection of patients for coronary angiography and coronary artery surgery should be based on the potential for benefit and should avoid "agism."     

Design of a trial evaluating myocardial cell protection with cariporide, an inhibitor of the transmembrane sodium-hydrogen exchanger: the Guard During Ischemia Against Necrosis (GUARDIAN) trial

Background  

Direct myocardial cell protection in patients with unstable angina or evolving myocardial infarction (MI) could prevent cell necrosis or reduce its extent, and minimize the risk of MI and death associated with percutaneous coronary interventions (PCIs) and coronary artery bypass surgery. The myocardial NHE plays a critical role in mediating the progression of ischemia to necrosis by promoting intracellular accumulation of sodium and calcium in exchange for hydrogen. Blockage of the system in various experimental models of ischemia and reperfusion had a strong antinecrotic effect. The present paper describes a trial that was intended to investigate the potential clinical benefit of cariporide, a potent and selective inhibitor of the NHE, in a large spectrum of at-risk patients.     

Anomalous origin of the left coronary artery from the pulmonary artery treated by palliative Vineberg operation in an infant

Anomalous origin of the left coronary artery from the pulmonary artery was diagnosed in an infant girl who had evidence of transmural myocardial infarction of the free wall of the left ventricle. At age 13 months, she underwent a palliative left Vineberg implant, and remained asymptomatic until she was 8 years of age. At that time, she underwent suturing of the left coronary ostium for obliteration of the left coronary shunt at the pulmonary artery. At age 13, she underwent aortocoronary bypass to the left main coronary artery, with end-to-end anastomosis. The patient remains asymptomatic to date. We believe that this is the first reported case of a Vineberg operation performed in an infant for palliation of an anomalous left coronary artery originating from the pulmonary artery. This method allows time for the development of collateral circulation to the left coronary artery before definitive surgery is performed.     

MYOCARDIAL INFARCTION FOLLOWING CAROTID ENDARTERECTOMY

Myocardial infarction has been the major cause of mortality following operation for cerebrovascular insufficiency. In our institution, a clinical diagnosis of coronary artery disease was made in 37 of 125 (29.6%) consecutive male patients having carotid endarterectomy. Six of these 37 patients developed postoperative myocardial infarction. In contrast, none of the 88 patients without coronary artery disease developed myocardial infarction. A more recently treated group of 20 patients who had undergone carotid artery surgery and had previously undergone coronary artery bypass for angina did not develop postoperative myocardial infarction. These data suggest that in patients with both coronary artery and carotid artery disease, prior or concomitant coronary artery bypass should be considered. Myocardial infarction has been the leading cause of early and late death following operation for cerebrovascular insufficiency.(1) DeBakey(2) found operative mortality in patients having surgery for cerebrovascular insufficiency directly related to the incidence of coronary artery disease. An increased operative mortality due to reinfarction has been found in patients recovering from recent myocardial infarction.(3) Cooley(4) found that in patients having aortocoronary bypass there was no increased operative mortality 30 days after myocardial infarction and this may apply to patients having carotid endarterectomy. Subendocardial postoperative infarction associated with minor T wave changes and slight enzyme elevation had a better prognosis than did transmural infarction causing significant Q waves, sequential ST and T wave changes and marked enzyme elevations.(5) The purpose of this study was to document our experience with myocardial infarction in patients undergoing carotid artery operation for clinical coronary artery disease. Consideration of the role of saphenous vein bypass in those patients with coronary artery disease was the background for this review even though the evidence that myocardial infarction can be prevented with saphenous vein bypass operation is only preliminary at the present time.(6)     

Advances in cell lineage reprogramming

As a milestone breakthrough of stem cell and regenerative medicine in recent years,somatic cell reprogramming has opened up new applications of regenerative medicine by breaking through the ethical shackles of embryonic stem cells.However,induced pluripotent stem(iPS) cells are prepared with a complicated protocol that results in a low reprogramming rate.To obtain differentiated target cells,iPS cells and embryonic stem cells still need to be induced using step-by-step procedures.The safety of induced target cells from iPS cells is currently a further concerning matter.More broadly conceived is lineage reprogramming that has been investigated since 1987.Adult stem cell plasticity,which triggered interest in stem cell research at the end of the last century,can also be included in the scope of lineage reprogramming.With the promotion of iPS cell research,lineage reprogramming is now considered as one of the most promising fields in regenerative medicine,will hopefully lead to customized,personalized therapeutic options for patients in the future.     

Capacities of multidetector spiral computed tomography in the planning and postoperative examination of patients undergoing coronary artery bypass surgery

Multiple investigations show that multidetector spiral computed tomography (MSCT) bypass grafting becomes an alternative to invasive coronary angiography in detecting coronary graft stenoses and occlusions. The investigation retrospectively estimated the patency of aortocoronary and mammary coronary artery anastomoses by MSCT bypass grafting. Examinations were made in 85 (326 anastomoses) patients who had undergone aortocoronary and mammary coronary artery bypass surgery and had MSCT bypass grafting within 3 years after the surgery. In the first year following the surgery, 18 patients with graft stenotic changes, as evidenced by MSCT, underwent intervention coronary angiography, the sensitivity and specificity of which was 100%. The results of clinical and instrumental examinations were also compared with graft incompetence, as shown on MSCT that revealed that MSCT bypass grafting was the only noninvasive technique to evaluate early coronary graft closure both in the absence of clear signs of myocardial ischemia according to the data of exercise tests and in the presence of recurrent angina pectoris.     

Adult-derived stem cells and their potential for use in tissue repair and molecular medicine

This report reviews three categories of precursor cells present within adults. The first category of precursor cell, the epiblast-like stem cell, has the potential of forming cells from all three embryonic germ layer lineages, e.g., ectoderm, mesoderm, and endoderm. The second category of precursor cell, the germ layer lineage stem cell, consists of three separate cells. Each of the three cells is committed to form cells limited to a specific embryonic germ layer lineage. Thus the second category consists of germ layer lineage ectodermal stem cells, germ layer lineage mesodermal stem cells, and germ layer lineage endodermal stem cells. The third category of precursor cells, progenitor cells, contains a multitude of cells. These cells are committed to form specific cell and tissue types and are the immediate precursors to the differentiated cells and tissues of the adult. The three categories of precursor cells can be readily isolated from adult tissues. They can be distinguished from each other based on their size, growth in cell culture, expressed genes, cell surface markers, and potential for differentiation. This report also discusses new findings. These findings include the karyotypic analysis of germ layer lineage stem cells; the appearance of dopaminergic neurons after implantation of naive adult pluripotent stem cells into a 6-hydroxydopamine-lesioned Parkinson's model; and the use of adult stem cells as transport mechanisms for exogenous genetic material. We conclude by discussing the potential roles of adult-derived precursor cells as building blocks for tissue repair and as delivery vehicles for molecular medicine.