摘要
干细胞分化产生新细胞，用来替代受损的心肌、瓣膜、血管及传导细胞，这种干细胞治疗方法潜力很大。最近随着心脏内多效祖细胞的发现及对多能的胚胎干细胞发育过程的了解，促进了可用于治疗心脏病的特异性细胞的开发。血循环中祖细胞分泌的细胞因子在受损部位浓集也有利于保护受损组织和新生血管形成。这些在基础科学中令人激动的发现还需要在动物模型上进行严格的验证以判断其未来在临床中的价值。
人们发现了干细胞的生物特性具有治疗人类疾病的巨大潜能，这一发现让科学家和公众都非常兴奋。动员组织内的内源性祖细胞或引入，诱导外源性干细胞分化以修复受损组织将对许多疾病的治疗产生巨大的影响，包括脑病，肌肉骨骼系统疾病，胰腺病及心脏病。尤其是心脏病，作为成人及儿童非感染性疾病中首位死亡原因，可以从再生治疗中获得很大益处。出生后心肌细胞似乎不再进入细胞周期进行分裂，因此在损伤后心脏几乎没有再生能力。长期以来，人们一直认为人类出生时有多少心肌细胞，死亡时仍是这些细胞。
但过去5年令人激动的新发现使我们重新评价心脏的保护治疗或再生治疗的可能性。与大脑相似，心脏似乎也充分储备着祖细胞，虽不能代替急性损伤时大量丢失的细胞，但可替换一生时间中缓慢凋亡而损失的细胞。在这里，我们对目前基础及临床科学中进展进行综述，这些发现将成为未来心肌再生治疗基础。
心脏及胚胎干细胞起源的祖细胞
在整个心肌中，心肌祖细胞（CPCs）可能以较低的基线水平不停地替代凋亡的心肌细胞。与最终分化成的心肌细胞不同，心肌祖细胞较小，不表达心肌细胞标志，并可以自行更新和增殖。在体外，有一些表面上有差异但又有共性祖细胞群（如Sca-1－, c-Kit-，Abcg2-的侧群细胞）可被诱导分化激活心肌细胞特异性的基因表达。在间充质干细胞中也能看到这种现象，但间充质干细胞似乎不能分化成功能完整的心肌细胞。在体内，Sca-1－, c-Kit-也可以分化成心肌细胞，参与急性心梗后受损心肌的修复，但它们的潜力有限，其原因之一是内源性心肌祖细胞数量很小。通过细胞因子动员并扩增内源性祖细胞的尝试为再生治疗提供了希望，但这种方法也存在争议。或许，内源性心肌祖细胞在对梗塞心脏末端进行有效修复前，其活性必须通过胚胎发育中正常祖细胞扩增和分化的机制被增强。
因为胚胎干细胞（ES）及祖细胞功能早期胚胎细胞相似，是不连续的细胞系，因此对心脏发生早期事件的阐明有助于对心肌祖细胞的了解和处理。多能干细胞的维持需要许多转炉调节因子，如NANOG,OCT4及SOX2。在一些关键的转录因子（包括MESP及T盒蛋白鼠短尾突变体表型）的作用下心肌祖细胞可直接分化成中胚层细胞系。接下来中胚层祖细胞的分化与胚胎发育中两个不同心脏发生区域的细胞相似，通常被称为第一和第二心脏区域，这些区域的细胞表达独特的祖细胞标志。如含LIM区的转录因子ISL1，它参与第二心脏区域细胞的分化，而含同源结构域的转录因子NKX2.5则是两个区域的共同标志。更有趣的是第二心脏区域残留的细胞不但可以分化为各种不同类型的细胞，即使在出生后还会持续存在。虽然不同亚群细胞系更精确的发育潜能还不清楚，但有潜能的心肌祖细胞还是可能不断分化成各种类型的细胞，包括心肌细胞，传导细胞，组成血管的细胞，持续参与心脏的维护。可见发现原始心脏区域的独特标志对于在出生后甚至是成年后定位干细胞是非常重要的。
第一和第二心脏区域的发育标志在为得到心肌祖细胞而对胚胎干细胞进行的富集过程中很有用。当细胞成簇生长成为胚胎体，小鼠和人的胚胎干细胞都能分化成许多细胞系，包括跳动的心肌细胞。有许多方法可以用来在此过程中增加心肌祖细胞的数量，包括加入维甲酸或二甲基亚砜，及抑制BMP的信号传递。调节WNT的信号传递可能也有用。除了关键的信号传递和转录过程可以直接决定心脏细胞系的发育外，还发现心肌特异性的微RNA（如miR-1和miR-133）参与了胚胎发育中心肌祖细胞的分化。它们可能也在启动和维持心肌发育过程中起作用。最后随着化学分子库及高通量筛选技术的进展已使筛选促进心肌祖细胞分化和增殖的小分子成为可能。
总之，许多研究都在对心肌祖细胞进行分离，并试图改变其生物学行为。这个过程是不可缺少的，因为直接将未分化的胚胎干细胞注入心脏会形成畸胎瘤。以上的方法为我们带来了希望，使我们能控制胚胎干细胞及非胚胎干细胞的增殖和分化，以驾驭这些细胞用于临床治疗。
其它类型的祖细胞
对干细胞基础生物学认识有进展的同时，临床研究也有一些结果引起争议。早先的研究认为小鼠骨髓起源的间充质干细胞（BMSCs）可分化成心肌细胞，因此心梗后注入间充质干细胞可使受损的心肌得到修复。最近有遗传学标志进行的研究表明间充质干细胞不能转化为心肌细胞，但它仍可能有有利的作用，可能是通过分泌旁分泌因子来保护心肌或促进血管发生。
在早期小鼠心肌形成的基础上，许多用自体骨髓间充质干细胞治疗急性心梗及缺血性心肌病的临床试验已开始。前期无对照的临床试验提示获益，但后续的随机试验结果不一致。几个小型研究报道在增加心脏泵血功能及冠脉灌流量，减少梗塞面积上有统计学意义，但至少最近一个更大规模的试验没有显示临床获益。同样，一个基于早期小鼠研究的随机临床试验，心梗后用集落刺激因子动员内源性骨髓间充质干细胞进行治疗的试验中也没看到心脏功能好转。
如果骨髓间充质干细胞确实能在生理学上产生益处，其机制也还不清楚。这使人们对现在还未完成的临床试验产生忧虑，这些试验都是基于骨髓间充质干细胞可以分化成心肌细胞的据说进行的。尽管现在的焦点已转移到骨髓间充质干细胞对缺氧心肌的非细胞自主效应上，仍有许多东西需要探索。干细胞分泌的血管生成因子及促进这些细胞存活的活化通路可能有助于缺氧心肌的保护和复苏，而减小组织损伤，改善心功能。如果旁分泌因子的确有明显作用，分离并高浓度给予这些因子或利用干细胞工程学的方法使间充质干细胞分泌大量因子可能会对心肌产生更强的保护。有趣的是，胸腺素β4（间充质干细胞有大量分泌）在急性心梗后可产生心脏保护作用，并可诱导小鼠的新生血管形成。未来，胸腺素β4及其它分泌因子的大规模动物及临床试验可能为今后的治疗提供希望，也许可避免缺氧性心肌病所需的细胞治疗。
前景
未来关于干细胞临床应用治疗心脏病的研究将以对细胞系分化认识的进展及对其向心脏血管细胞分化及多能性的调节为基础。用再生的方法修复受损的心肌或对心肌细胞系某一亚群缺乏的儿童进行治疗都会遇到一些困难。我们不但要掌握能诱导，扩增干细胞使之分化为心肌细胞系的技术，还要知道如何避免它们分化成为对心脏内环境有危害的细胞。正确的输注方法，可以用来避免并发症及可引起心律失常的异常电耦合，这种情况曾在将骨骼肌移植到心脏时发生过。最后，如果是非自体的干细胞移植，还要解决周围组织的免疫排斥问题。在这方面，通过体细胞核转导技术及细胞融合技术产生出个体特异性的干细胞系可使经生物工程处理过的干细胞拥有自己的遗传物质，很方便的应用于临床治疗及药理学研究。
Nature 441, 1097-1099(29 June 2006) | doi:10.1038/nature04961; Published online 28 June 2006
Potential of stem-cell-based therapies for heart disease
Deepak Srivastava1 and Kathryn N. Ivey1
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Abstract
The use of stem cells to generate replacement cells for damaged heart muscle, valves, vessels and conduction cells holds great potential. Recent identification of multipotent progenitor cells in the heart and improved understanding of developmental processes relevant to pluripotent embryonic stem cells may facilitate the generation of specific types of cell that can be used to treat human heart disease. Secreted factors from circulating progenitor cells that localize to sites of damage may also be useful for tissue protection or neovascularization. The exciting discoveries in basic science will require rigorous testing in animal models to determine those most worthy of future clinical trials.
Rarely has anything so energized scientists and the lay public alike as the enormous potential of stem-cell biology to treat human disease. The ability to mobilize endogenous progenitor cells in organs or to introduce and differentiate exogenous stem cells for tissue repair could have an impact on many diseases, including those affecting the brain, skeletal muscle, pancreas and heart. Regenerative therapies could be particularly beneficial for heart disease — the number one killer in adults and the leading non-infectious cause of death in children1. Cardiomyocytes do not seem to enter the cell cycle after birth, and consequently the heart has almost no regenerative capacity after injury. The long-held dogma has been that the heart cells with which you are born are the ones with which you die.
However, exciting new findings in the past 5 years have caused us to re-evaluate the potential of protective or regenerative cardiac therapies. Like the brain, the heart seems to have reservoirs of progenitor cells that may not be sufficient to replace the acute loss of a large number of cells, but may be able to replace a slow apoptotic loss of cells over a lifetime. Here, we examine the current basic and clinical science that is forming the foundation of future approaches to cardiac regeneration.
Cardiac and ES-cell-derived progenitor cells
Throughout the myocardium, pools of cardiac progenitor cells (CPCs) may participate in the continual replacement of apoptotic cardiomyocytes at a low basal level. Unlike terminally differentiated cardiac cells, CPCs are small cells that do not express cardiac markers and that can self-renew and proliferate. Several seemingly different but overlapping populations of progenitor cells (such as Sca-1- (ref. 2), c-Kit- (ref. 3) and Abcg2- (ref. 4) expressing side populations) can be induced to activate cardiomyocyte-specific genes in vitro; however, this effect has been also observed in mesenchymal stem cells, which do not fully differentiate into functional heart cells5. In addition, Sca1- or c-Kit-expressing cells may differentiate into cardiomyocytes in vivo, contributing to repair of the damaged heart after acute myocardial infarction, but their potential is limited, in part, by the small number of endogenous CPCs. Attempts to mobilize and to expand endogenous progenitor cells by introducing growth factors hold promise but remain controversial6. It is likely that the activity of endogenous CPCs will have to be augmented, through knowledge of the mechanisms of normal progenitor expansion and determination during embryonic development, before these cells will contribute substantially in the extreme setting of infarcted hearts.
Because embryonic stem (ES) and progenitor cells resemble early fetal cells that are adopting discrete lineages, elucidating the early developmental events of cardiogenesis has been instructive for understanding and manipulating CPCs. Pluripotent stem cells maintained through transcriptional regulators (such as NANOG, OCT4 and SOX2; ref. 7) are directed to differentiate into the mesoderm lineage by key transcription factors, including MESP and the T-box protein brachyury8,9 (Fig. 1). Subsequent determination of mesoderm progenitors mimics the embryonic developmental potential of two distinct fields of cells that give rise to the heart. Often referred to as the first and second heart fields, cells in these regions express unique markers of progenitor cells10. For example, the LIM-domain-containing transcription factor islet1 (ISL1) is involved in the differentiation of second heart field cells11, whereas the homeodomain-containing transcription factor NKX2.5 is a marker of both heart fields10. Most interestingly, remnant second heart field cells may not only be able to differentiate into many cell types, but also persist in the postnatal heart12 (Fig. 1). The pool of potential CPCs might be involved in continual maintenance of the heart by differentiating into several types of cardiac cell, including muscle, conduction and vascular cells, although the precise lineage potential of distinct subtypes remains to be determined (Fig. 2). It will be important to identify specific markers of the primary heart field to locate progenitor cells postnatally and even into adulthood.
Developmental markers of the primary and secondary heart fields may be useful in enriching ES cells for CPCs. When allowed to grow in clusters called 'embryoid bodies', mouse and human ES cells can differentiate into many lineages, including beating cardiomyocytes. Numerous approaches to increase the number of CPCs in this system have been devised, including the addition of retinoic acid or dimethyl sulphoxide, and the inhibition of BMP signalling13. Modulation of WNT signalling may also be useful14. In addition to key signalling and transcriptional events that may direct the cardiac lineage, the discovery of microRNAs that are muscle-specific (for example, miR-1 and miR-133) and involved in differentiation of CPCs in the embryo15,16 raises the interesting possibility that microRNAs could be useful in initiating or sustaining the cardiogenic programme. Finally, improvements in chemical libraries and high-throughput screens make it possible to screen for small molecules that could regulate the proliferation or differentiation of CPCs17.
In summary, numerous studies are underway to isolate and modify the behaviour of CPCs. Such pursuits are essential because the direct introduction of undifferentiated ES cells into the heart results in the formation of teratomas18. The different lines of investigation and approach described above provide hope that we may be able to regulate the commitment, proliferation and differentiation of ES or non-ES cells into cardiomyocytes, and to harness these cells for therapeutic purposes.
Other types of progenitor cell
Advances in understanding the basic biology of stem cells have been balanced by mixed and controversial results from translational and clinical studies. Early studies in mice suggested that bone-marrow-derived mesenchymal stem cells (BMSCs) could differentiate into cardiomyocytes19, and thus the introduction of BMSCs after myocardial infarction might induce the repair of damaged myocardium. More recent studies with genetically marked cells indicate that BMSCs do not transdifferentiate into cardiomyocytes20,21,22. It remains possible that BMSCs do confer some beneficial effects, possibly by secreting paracrine factors that are cardioprotective or angiogenic23.
On the basis of early mouse work suggesting myogenesis, numerous clinical trials with autologous BMSCs were begun in individuals with acute myocardial infarction or ischaemic myocardium24. Early anecdotal reports suggested some benefit, but subsequent randomized trials yielded mixed results. Several small studies reported a statistically significant increase in cardiac pumping ability and coronary perfusion, and a decrease in infarct size. At least one larger recent trial, however, has not shown a clinical benefit25. Similarly, a randomized trial based on earlier mouse data26, in which granulocyte colony-stimulating factor was used to mobilize endogenous BMSCs after infarction, did not show improvement in cardiac function27.
If BMSCs do confer physiological benefit, then they do so by an unknown mechanism, raising concern for ongoing or pending clinical trials, many of which initially assumed that BMSCs can differentiate into new cardiomyocytes. Although the focus has now shifted to understanding the potential non-cell-autonomous effects of BMSCs on hypoxic myocardium, much remains to be learned. Secreted angiogenic factors and/or activation of pathways that promote cell survival might protect and rescue hypoxic myocardium, thereby limiting damage to tissue and improving cardiac function23. If paracrine factors are the key agents, isolating and delivering such factors at high concentrations or engineering BMSCs to secrete larger amounts could result in more significant protection28. Interestingly, thymosin 4, which is secreted in very large quantities by BMSCs28,29, is cardioprotective after acute myocardial infarction30 and induces angiogenesis in mice31,32. Future large-animal and clinical trials of thymosin 4 and other secreted factors hold promise and may obviate the need for cell-based therapy for the at-risk hypoxic myocardium.
The road ahead
Future research on the clinical applications of stem cell biology for human heart disease will be based on advances in understanding cell lineage decisions and the regulation of pluripotency and differentiation in cardiac and vascular cells. Approaches for regenerating damaged muscle and for treating children who lack specific subsets of cardiac lineages will face many hurdles. The ability not only to guide and expand stem cells into the cardiac lineage but also to repress alternative fates will be crucial to avoid differentiation into cell types that may be harmful to cardiac homeostasis. Methods for safe delivery, migration and proper integration of stem cells will need to be perfected to avoid complications and abnormal electrical coupling that could lead to arrhythmias, as has been experienced with introduction of skeletal muscle into the myocardium33. Finally, it will be essential to solve the immunological issues surrounding rejection if non-autologous sources of stem cells are used. In this respect, technologies to develop individual-specific stem-cell lines through somatic-cell nuclear transfer34 or cell fusion35 may allow engineered stem cells containing the individual's own genetic material to be used both for treatment and for studies of pharmacological efficacy.
编辑：蓝色幻想
