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

描述(由申请人提供)： 人脐血(UCB)中含有的间充质干细胞(MSCs)比成人骨髓间充质干细胞(BM-MSCs)具有更高的多潜能。然而，这些细胞一直很难获得，因为脐带血中的MSCs数量非常少(1×108个单核细胞中只有5到30个)。到目前为止，骨髓间充质干细胞的分离依赖于它们的塑料黏附能力。UCB中大多数极不成熟的MSCs可能会被忽略，因为它们附着在塑料上的能力很差。我们以前的研究表明，骨髓细胞形成的细胞外基质(ECM)促进了MSC的附着和增殖，并保持了其干细胞特性(Chen等，2007，JBMR，22：1943)。使用这个系统，我们发现人脐血中可能含有大量的MSCs，它们附着在ECM上(至少是之前报道的10,000-100,000倍)，但不附着在塑料上。更重要的是，将通过ECM黏附获得的脐带血来源的间充质干细胞(UCB-MSCs)植入免疫缺陷小鼠体内，产生了3种胚胎生殖层来源的组织，包括骨、肌肉、脂肪、腺、肠和神经纤维。受到这些发现的鼓舞，我们提出假设，人类脐带血中含有大量的胚胎样干细胞，这些干细胞一般有可能用于组织再生，特别是心肌重建。为了验证这一假设，将追求以下三个具体目标：具体目标1利用基因芯片技术，确定人胚胎干细胞(HES细胞)、ECM粘附法分离的UCB-MSCs和塑料粘附法分离的UCB-MSCs以及人成年BM-MSCs之间基因表达的异同。目的2研究体外细胞外基质黏附获得的人脐血-间充质干细胞在成骨细胞、脂肪细胞、软骨母细胞和心肌细胞(中胚层)、神经细胞(外胚层)和肝细胞(内胚层)等特定细胞系的诱导下，体外选择性分化为所需细胞系的能力。具体目标3是利用已建立的小鼠模型评价通过ECM黏附获得的移植UCB-MSCs改善心肌梗死(MI)后心功能的能力。尽管HES细胞具有巨大的发展潜力，但人们似乎普遍认为，一种不那么关键的细胞材料来源更适合于组织再生的临床应用。如果所提出的研究证实ECM可以促进从脐带血中提取大量的胚胎样干细胞，那么由此产生的高功能的脐带血来源的MSCs将使其成为HES细胞的替代细胞用于临床应用是可行的。特别是，这些研究的最终结果可能是一种高度实用的基于干细胞的疗法来治疗心肌梗塞后的退伍军人。

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.