Use of umbilical cord as a unique stem cell source for tissue regeneration

描述（由申请人提供）： 人脐带血（UCB）含有间充质干细胞（MSC），其具有比成人骨髓源性MSC（BM-MSC）更高的多向分化潜能。然而，这些细胞一直难以获得，因为UCB中MSC的数量极低（1 x 108个单核细胞中约5至30个）。迄今为止，MSC的分离依赖于其塑性粘附能力。UCB中大多数极不成熟的MSC可能会被遗漏，因为它们粘附塑料的能力很差。我们先前的研究表明，由骨髓细胞产生的细胞外基质（ECM）增强MSC附着和增殖，并保留其干细胞特性（Chen等人，2007，JBMR，22：1943）。使用这个系统，我们发现人UCB可能含有大量的MSC，这些MSC粘附在ECM上（比以前报道的至少高10，000到100，000倍），但不粘附在塑料上。更重要的是，将通过ECM粘附获得的UCB-derived MSCs（UCB-MSCs）植入免疫功能低下的小鼠体内，产生了3种胚胎胚层来源的组织，包括骨、肌肉、脂肪、腺体、肠和神经纤维。受这些发现的鼓舞，我们提出了这样的假设，即人脐带血中含有大量的胚胎样干细胞，这些干细胞有可能用于组织再生，特别是心肌重建。为了检验这一假设，将追求以下3个具体目标：具体目标1是使用微阵列技术确定人胚胎干细胞（hES细胞）、通过ECM粘附分离的UCB-MSC与通过塑料粘附分离的UCB-MSC以及人成人BM-MSC之间的整体基因表达的相似性和差异。具体目的2是确定通过ECM粘附获得的人UCB-MSC在已知诱导定型为特定细胞谱系的条件下体外选择性分化为源自3个胚胎胚层的所需细胞谱系的能力，所述细胞谱系包括成骨细胞、脂肪细胞、成软骨细胞和心肌细胞（中胚层）、神经（外胚层）和肝细胞（内胚层）。具体目的3是使用良好建立的小鼠模型评估通过ECM粘附获得的移植UCB-MSC改善心肌梗死（MI）后心脏功能的能力。 尽管hES细胞具有巨大的发育潜力，但似乎普遍认为，对于组织再生的临床应用而言，较不重要的细胞材料来源将是优选的。如果所提出的研究证实ECM可以增强从UCB中回收大量胚胎样干细胞，那么由此产生的高功能UCB衍生的MSC的无限来源将使其成为基于细胞的临床应用的hES细胞的替代品成为可行的。特别是，这些研究的最终结果可能是一个非常实用的基于干细胞的治疗心肌梗死后退伍军人。 公共卫生相关性： 随着美国人口的老龄化，美国的退伍军人也在老龄化。根据2003年全国退伍军人健康和对退伍军人管理局的依赖调查，47%的退伍军人年龄在65岁或以上。心肌梗死（MI）作为一种与年龄相关的疾病，是美国心力衰竭和死亡的主要原因之一。目前，在大面积MI后完全恢复心脏功能的最佳选择是心脏移植。然而，它受到供体可用性和移植排斥的限制。最近，通过注射干细胞再生梗死心肌已被提出作为一种替代疗法。如果所提出的研究证实脐带血含有大量的胚胎样干细胞，其可以通过ECM粘附程序分离，并且可以在体外选择性分化为心肌细胞并在MI后在体内重建心肌，则将促进开发用于治疗MI后退伍军人的基于细胞的再生的新治疗方法。

DESCRIPTION (provided by applicant): Human umbilical cord blood (UCB) contains mesenchymal stem cells (MSCs) that have higher multipotentiality than adult marrow-derived MSCs (BM-MSCs). However, these cells have been difficult to obtain because the number of MSCs in UCB is extremely low (~5 to 30 out of 1 x 108 mononuclear cells). To date, the isolation of MSCs has depended upon their plastic-adhesion capacity. Most extremely immature MSCs in UCB are likely missed because their ability to adhere to plastic is poor. Our previous studies demonstrated that cellular extracellular matrix (ECM) made by bone marrow cells enhanced MSC attachment and proliferation, and retained their stem cell properties (Chen, et al, 2007, JBMR, 22:1943). Using this system, we found that human UCB may contain a large number of MSCs that adhere to the ECM (at least 10,000- to 100,000-fold greater than that previously reported), but not to plastic. More importantly, implantation of UCB-derived MSCs (UCB-MSCs) obtained by ECM adhesion into immunocompromised mice generated 3 embryonic germ layers-derived tissues including bone, muscle, fat, gland, intestine and nerve fibers. Encouraged by these findings, we propose the hypothesis that human UCB contains a large number of embryonic-like stem cells that have the potential to be used for tissue regeneration in general and myocardial reconstruction in particular. To test this hypothesis, the following 3 Specific Aims will be pursued: Specific Aim 1 is to determine the similarities and differences in global gene expression among human embryonic stem cells (hES cells), UCB-MSCs isolated by ECM adhesion versus those isolated by plastic adhesion, and human adult BM-MSCs, using microarray technology. Specific Aim 2 is to determine the ability of human UCB-MSCs obtained by ECM adhesion to selectively differentiate into desired cell lineages originated from 3 embryonic germ layers in vitro under conditions known to induce commitment to a specific cell lineage, including osteoblasts, adipocytes, chondroblasts and cardiomyocytes (mesoderm), nerve (ectoderm) and hepatocytes (endoderm). Specific Aim 3 is to evaluate the capability of transplanted UCB-MSCs obtained by the ECM adhesion to improve heart function after myocardial infarction (MI) using a well-established mouse model. Despite the great developmental potential of hES cells, there appears to be widespread agreement that a less critical source of cellular material would be preferable for the clinical use of tissue regeneration. If the proposed studies confirm that ECM can enhance retrieval of large numbers of embryonic-like stem cells from UCB, the resulting unlimited source of highly functional UCB-derived MSCs would make it feasible to be alternative to hES cells for cell-based clinical application. Specially, the ultimate outcome of these studies may be a highly practical stem cell- based therapy to treat the post-MI veteran. PUBLIC HEALTH RELEVANCE: As the US population is aging, so too is the US veteran population. According to the 2003 National Survey of Veteran Enrollees' Health and Reliance on VA, 47% of veterans were 65 years or older. Myocardial infarction (MI), as an age-related disease, is one of the leading causes of heart failure and death in the US. Currently, the best option for completely restoring cardiac function after a large MI is heart transplantation. However, it is limited by donor availability and transplant rejection. Recently, regeneration of infracted myocardium by injecting stem cells has been proposed as an alternative therapy. If the proposed studies confirm that umbilical cord blood contains a large number of embryonic- like stem cells that can be isolated by ECM adhesion procedures, and that can selectively differentiate into cardiomyocytes in vitro and reconstruct myocardium in vivo after MI, it will facilitate development of novel therapeutic approaches for cell-based regeneration to treat post-MI veteran.