Multipotent Stem Cells in Post Natal Bone Marrow

描述（由申请人提供）：干细胞是一种具有广泛 增殖潜力，可以分化成几种细胞类型。的 典型的干细胞是胚胎干（ES）细胞，其具有无限的 自我更新和多能分化潜能。组织特异性干 细胞具有较少的自我更新能力并且不是多能的。一些 最近的研究表明，组织特异性干细胞可能具有以下能力： 从不相关的器官中产生组织细胞。无论这是意料之外的 可塑性构成了“转分化”，或者一小群 多能干细胞在出生后组织中持续存在尚不清楚 干细胞存在于出生后的组织中， 增殖和分化潜力开辟了使用 自体干细胞治疗退行性、创伤性或先天性 疾病除了使用自体干细胞的免疫优势外 组织来源的干细胞也不受伦理道德的限制。 注意事项。我们鉴定了人（h）、鼠（m）和大鼠（r）中的细胞， 出生后骨髓（BM），在单细胞水平上， 分化和广泛的增殖潜力。我们将其命名为 多能成体祖细胞或MAPC。(A)hMAPC、mMAPC和rMAPC 体外分化中胚层细胞类型，具有神经外胚层的细胞， 具有内胚层特征。(B)使用逆转录病毒标记和评估 整合子插入位点，我们已经显示hMAPC多谱系 分化来源于单细胞。(C)hMAPC、mMAPC和rMA.PC 表达有活性的端粒酶，可进行90+细胞倍增，无端粒 缩短，表明它们不会衰老。(D)hMAPC。mMAPC和rMAPC 表达oct-4、Rex-1 mRNA和用Ab染色的培养的hMAPC部分， SSEA-4。(E)我们有证据表明MAPC在体内植入并持续2+ 即使分化的移植细胞的单细胞起源 在多个组织中的特异性细胞尚未得到证实。(F)类似细胞 具有广泛的增殖和多系分化潜能， 在老鼠的大脑和肌肉中发现。因为含有MAPC的培养物仍然 异质性，我们计划进一步表征的表型和基因型， MAPC。我们还将检验MAPC可能存在于其他器官中的假设， 来源于不同器官的MAPC的表型相似。这 将在以下3个目标中完成：SA 1：定义细胞表面 MAPC的表型特征和表达基因谱。如已经 对于其他干细胞，我们认为MAPC可以从新鲜的 基于细胞表面或功能（例如静止），将组织纯化至高纯度 特色MAPC的表达基因谱将使我们能够 区分MAPC和组织定向干细胞，如MSC。我们也 有初步证据表明MAPC可能存在于BM和计划以外的组织中 进一步鉴定小鼠其他器官中MAPC的性质。SA 2：进一步定义 MAPC的增长需求。我们将测试自分泌因子是否 体外最佳MAPC生长所必需的，以及是否存在更多的 定向祖细胞对MAPC培养的同质性产生负面影响。 SA 3：确定mMAPC的体内分化能力。最终证明， MAPC是真正的干细胞将需要我们证明长期植入 并在克隆期分化为中胚层和非中胚层细胞类型 体内水平。我们将测试MAPC是否在体内分化为组织 以及当器官受损时这种情况是否会增强。

DESCRIPTION (provided by applicant): A stem cell is a cell with extensive proliferation potential that can differentiate into several cell types. The quintessential stem cell is the embryonal stem (ES) cell which has unlimited self renewal and multipotent differentiation potential. Tissue specific stem cells have less self-renewal ability and are not multipotent. A number of recent studies suggest that tissue specific stem cells may have the ability to generate cells of tissues from unrelated organs. Whether this unexpected plasticity constitutes "transdifferentiation" or whether a small population of multipotent stem cells persists in post-natal tissues is not known The finding that stem cells exist in post-natal tissues with previously unknown proliferation and differentiation potential opens up the possibility of using autologous stem cells to treat a host of degenerative, traumatic or congenital diseases. Aside from the immunological advantage of using autologous stem cells, tissue derived stem cells are also not encumbered by ethical considerations. We identified cells in human (h), murine (m) and rat (r), post-natal bone marrow (BM) that have, at the single cell level, multipotent differentiation and extensive proliferation potential. which we named Multipotent Adult Progenitor Cell or MAPC. (A) hMAPC, mMAPC and rMAPC differentiate in vitro mesodermal cell types, cells with neuroectodermal and with endodermal features. (B) Using retroviral marking and evaluation of the integrant insertion site, we have shown for hMAPC that multi-lineage differentiation is derived from single cells. (C) hMAPC, mMAPC and rMA.PC express active telomerase and can undergo 90+ cell doublings without telomere shortening, suggesting that they do not senesce. (D) hMAPC. mMAPC and rMAPC express oct-4, Rex-i mRNA and a fraction of cultured hMAPC stain with the Ab, SSEA-4. (E) We have evidence that MAPC engraft in vivo and persist for 2+ months even though single cell origin of the engrafted cells that differentiate in multiple tissue specific cells has not yet been proven. (F) Similar cells with extensive proliferative and multilineage differentiation potential can be found in murine brain and muscle. Because MAPC containing cultures are still heterogeneous, we plan to further characterize the phenotype and genotype of MAPC. We will also test the hypothesis that MAPC may exist in other organs but that the phenotype of MAPC derived from different organs will be similar. This will be accomplished in the following 3 aims: SA1: To define the cell-surface phenotypic characteristics and expressed gene profile of MAPC. As has been shown for other stem cells, we believe that MAPC can be selected from fresh tissue to high purity based on cell surface or functional (e.g. quiescence) characteristics. The expressed gene profile of MAPC will allow us to discriminate between MAPC and tissue committed stem cells, such as MSC. We also have preliminary evidence that MAPC may exist in tissues other than BM and plan to further characterize MAPC from other organs in mouse. SA2: To further define the growth requirements of MAPC. We will test if autocrine factors are necessary for optimal MAPC growth in vitro, and whether presence of more committed progenitors negatively impacts on culture of MAPC to homogeneity. SA3: To define the in vivo differentiation capacity of mMAPC. Final proof that MAPC are true stem cells will require that we demonstrate long-term engraftment and differentiation into mesodermal and non-mesodermal cell types at the clonal level in vivo. We will test whether MAPC differentiate in vivo to tissue specific cells and whether this is enhanced when organs are damaged.