Isolation, characterization, and transplantation of candidate stem cells

候选干细胞的分离、表征和移植

基本信息

批准号：
7593477
负责人：
John Tisdale
金额：
$ 32.01万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7593477
关键词：
ABCG2 gene ATP-Binding Cassette Transporters Adult Affect Albumins Autoimmunity Benign Beta Cell Bone Marrow Bone Marrow Cells Bone Marrow Transplantation CD34 Antigens CYP2B6 gene Carbon Cell Nucleus Cells Chloride Ion Chlorides Clinical Culture Media Cultured Cells Cytochrome P450 Cytokeratin 18 Cytokeratin 8 Cytoplasm Cytoplasmic Granules Disease Dose Dyes Flow Cytometry Gene Expression Profile HOE 33342 Hematopoiesis Hematopoietic Hematopoietic stem cells Hepatic Hepatocyte Human Insulin Insulin-Dependent Diabetes Mellitus Knowledge Lead Life Liver Liver Function Tests Malignant - descriptor Methods Microarray Analysis Modeling Molecular Mus Natural regeneration Numbers Organ Pancreas Phenotype Population Process Protein C Inhibitor Reverse Transcriptase Polymerase Chain Reaction SCID Mice Side Source Speed Staining method Stains Stem cells Stress Techniques Testing Transplantation Viral Vector accelerator mass spectrometry base clinical application human disease in vivo irradiation reconstitution repaired success transcription factor transdifferentiation

项目摘要

Hematopoietic stem cells reside within the bone marrow post-natally, providing all hematopoietic lineages for the life of the host, and their extensive characterization over the last several decades has led to clinical application including bone marrow transplantation for benign and malignant disorders affecting the hematopoietic compartment. Isolation of bone marrow cells based upon expression of the CD34 antigen enriches for the primitive compartment, and techniques to isolate the CD34-positive population are commonly used in clinical practice. Recently, a method for the isolation of murine bone marrow cells capable of reconstituting hematopoiesis after lethal irradiation based solely upon dual wavelength flow cytometry after staining with the vital dye, Hoechst 33342, was described by Goodell et. al. The molecular basis of these side population cells was recently attributed to expression of the ABC transporter, ABCG2. Side population (SP) cells are highly enriched for hematopoietic repopulating ability, with one cell capable of reconstituting hematopoiesis in irradiated recipient mice. We reasoned that this phenotype is conserved among organs with the capacity for post-natal regeneration. We have recently isolated SP cells from the non-parynchymal portion of human cadaveric liver and have cultured these cells in hepatic culture media. Hepatic SP cells generated colonies of cells demonstrating granule rich cytoplasm and dense, often double nuclei consistent with hepatocytes. These hepatocyte-like cells expressed markers of human hepatocytes including HepPar, cytokeratin-8, and human albumin. RT-PCR confirmed the expression of hepatocyte markers including albumin, cytokeratin-18, along with the more specific markers, 1 antitrypsin and the human P450 gene, CYP2B6. We have now developed an in vivo rescue model in NOD/SCID mice treated with sublethal dosing of carbon-tetra-chloride, and initial results suggest that SP cells are capable of affecting a normalization of liver function test similar to mature hepatocytes when compared to controls. Further in vivo characterization of these SP cells is ongoing. Additionally, SP cells have been isolated from the adult pancreas, and microarray analysis demonstrates gene expression profile similar to that of bone marrow derived SP cells. Initial attempts to differentiate pancreatic SP cells toward a mature beta-cell phenotype have met with only limited success and these studies are ongoing, yet their quiescence in culture raises the question whether beta-cells can regenerate in adult hosts. We have thus developed a method for assessing beta cell regeneration post-natally using 14C dating by accelerator mass spectrometry. Initial results suggest limited turnover, and the study is ongoing. Finally, in an attempt to generate insulin producing cells that could resist the autoimmunity that characterizes Type I diabetes, we have tested whether the transfer of several transcription factors via viral vectors in vivo could lead to hepatocyte-to-beta cell transdifferentiation, with evidence that this process occurs a low level if at all.

造血干细胞在后血压下驻留在骨髓内，为宿主的生命提供了所有造血谱系，并且它们在过去几十年中的广泛特征导致了临床应用，包括骨髓移植的良性和恶性疾病，影响造血室。基于CD34抗原富集的原始区室的表达，骨髓细胞的分离，分离CD34阳性种群的技术通常用于临床实践中。最近，Goodell et介绍了一种在致命照射后分离能够在致命照射后重新构成造血的方法，该方法仅基于双波长流式细胞仪，用重要的染料染色后，Hoechst 33342，由Goodell et染色。 al。这些侧种群细胞的分子基础最近归因于ABC转运蛋白ABCG2的表达。侧种群（SP）细胞高度富集造血重塑性能力，一个细胞能够在受辐照的受体小鼠中重构造血。我们认为，这种表型在具有产后再生能力的器官中是保守的。最近，我们从人尸体肝的非核酸一部分中分离出SP细胞，并在肝培养基中培养了这些细胞。肝SP细胞产生的细胞菌落表现出富含颗粒的细胞质和致密，通常与肝细胞一致。这些类似肝细胞的细胞表达了人类肝细胞的标志物，包括Heppar，Cytokeratin-8和人白蛋白。 RT-PCR证实了包括白蛋白，Cytokeratin-18在内的肝细胞标记的表达，以及更具体的标记，1抗胰蛋白酶和人P450基因CYP2B6。现在，我们已经在接受碳 - 氯化碳 - 氯化碳剂量治疗的点数/SCID小鼠中开发了一种体内救援模型，并且初步结果表明，与对照组相比，SP细胞能够影响类似于成熟的肝细胞的肝功能测试的归一化。这些SP细胞的进一步体内表征正在进行中。另外，已经从成年胰腺中分离出SP细胞，微阵列分析表明基因表达谱类似于骨髓衍生的SP细胞。将胰腺SP细胞分化为成熟的β细胞表型的最初尝试仅取得了有限的成功，这些研究正在进行中，但是它们在培养中的静止提出了一个疑问，质疑β细胞在成年宿主中是否可以再生。因此，我们已经开发了一种使用加速器质谱法使用14C日期来评估β细胞再生的方法。最初的结果表明营业额有限，并且该研究正在进行中。最后，为了产生可以抵抗I型糖尿病的自身免疫性的胰岛素细胞，我们测试了是否通过体内病毒向量转移多个转录因子的转移是否会导致肝细胞到甲基甲虫细胞的差异，并有证据表明该过程在所有水平上都在较低水平上发生。