Multipotent Stem Cells in Post Natal Bone Marrow

描述(申请人提供)：干细胞是一种广泛存在的细胞 可分化为几种细胞类型的增殖潜能。这个 最典型的干细胞是胚胎干细胞，它具有无限的 自我更新和多潜能分化潜能。组织特异性干细胞 细胞自我更新能力较弱，不具备多能性。一批 最近的研究表明，组织特异性干细胞可能具有 从无关的器官中产生组织细胞。这是不是出乎意料的 可塑性构成了“转分化”，或者是一小部分人 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组织中，这是以前未知的 增殖和分化潜能为利用 自体干细胞治疗多种退行性、创伤性或先天性 疾病。除了使用自体干细胞的免疫学优势外 细胞，组织来源的干细胞也不受伦理道德的束缚 考虑因素。我们鉴定了人(H)、小鼠(M)和大鼠(R)的细胞， 出生后骨髓(BM)在单细胞水平上具有多潜能 分化能力和广泛的增殖潜能。我们把它命名为 多能成体祖细胞或MAPC。(A)hMAPC、mMAPC和rMAPC 在体外分化中胚层细胞类型、具有神经外胚层的细胞和 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，我们已经显示了hMAPC的多谱系 分化来源于单个细胞。(C)hMAPC、mMAPC和rMA.PC 表达活性端粒酶，可在无端粒的情况下进行90细胞倍增 变短，表明它们不会衰老。(D)hMAPC。MMAPC和rMAPC 用该抗体表达Oct-4、Rex-I和部分培养的hMAPC染色， SSEA-4。(E)我们有证据表明MAPC在体内植入并持续2 几个月，即使是分化的移植细胞的单一细胞来源 在多个组织特异性细胞中的作用尚未得到证实。(F)相似牢房 具有广泛的增殖和多系分化潜能 在小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表型和基因型。 MAPC。我们还将测试MAPC可能存在于其他器官的假设，但 来自不同器官的MAPC表型将是相似的。这 将通过以下3个目标实现：SA1：定义细胞表面 MAPC的表型特征和表达基因图谱。一如既往 对于其他干细胞，我们认为MAPC可以从新鲜细胞中挑选出来 基于细胞表面或功能的高纯度组织(如静止期) 特点。MAPC的表达基因图谱将使我们能够 区分MAPC和组织承诺干细胞，如MSC。我们也 有初步证据表明MAPC可能存在于BM和PLAN以外的组织中 进一步鉴定小鼠体内其他器官的MAPC。SA2：进一步定义 MAPC的生长要求。我们将测试自分泌因子是否 MAPC在体外最佳生长所必需的，以及是否存在更多 承诺的祖细胞对MAPC的培养产生负面影响，使其同质性。 SA3：确定mMAPC在体内的分化能力。最后的证据是 MAPC是真正的干细胞将需要我们展示长期植入 克隆时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是否在体内向组织分化 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.