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.

造血干细胞出生后存在于骨髓中，为宿主的一生提供所有造血谱系，过去几十年来，它们的广泛表征已导致临床应用，包括骨髓移植治疗影响造血室的良性和恶性疾病。基于 CD34 抗原表达的骨髓细胞分离可富集原始区室，并且分离 CD34 阳性群体的技术在临床实践中常用。最近，Goodell 等人描述了一种分离小鼠骨髓细胞的方法，该方法仅基于用活体染料 Hoechst 33342 染色后的双波长流式细胞术，在致死照射后能够重建造血功能。等人。这些侧群细胞的分子基础最近归因于 ABC 转运蛋白 ABCG2 的表达。侧群 (SP) 细胞高度富集造血再生能力，其中一种细胞能够在受辐射的受体小鼠中重建造血功能。我们推断这种表型在具有产后再生能力的器官中是保守的。我们最近从人尸体肝脏的非实质部分分离出 SP 细胞，并在肝培养基中培养这些细胞。肝SP细胞产生细胞集落，表现出富含颗粒的细胞质和致密的、通常是与肝细胞一致的双核。这些肝细胞样细胞表达人类肝细胞的标记物，包括 HepPar、细胞角蛋白-8 和人白蛋白。 RT-PCR 证实了肝细胞标记物的表达，包括白蛋白、细胞角蛋白-18，以及更特异性的标记物 α1 抗胰蛋白酶和人类 P450 基因 CYP2B6。我们现已在接受亚致死剂量四氯化碳治疗的 NOD/SCID 小鼠中开发了体内救援模型，初步结果表明，与对照相比，SP 细胞能够影响肝功能测试的正常化，类似于成熟肝细胞。这些 SP 细胞的进一步体内表征正在进行中。此外，已从成体胰腺中分离出 SP 细胞，微阵列分析表明其基因表达谱与骨髓来源的 SP 细胞相似。将胰腺 SP 细胞分化为成熟 β 细胞表型的初步尝试仅取得了有限的成功，这些研究仍在进行中，但它们在培养物中的静止状态提出了 β 细胞是否可以在成年宿主中再生的问题。因此，我们开发了一种利用加速器质谱法进行 14C 测年来评估出生后 β 细胞再生的方法。初步结果表明营业额有限，研究仍在进行中。最后，为了尝试产生能够抵抗 I 型糖尿病特征的自身免疫的胰岛素产生细胞，我们测试了通过体内病毒载体转移几种转录因子是否可以导致肝细胞向 β 细胞的转分化，有证据表明如果有的话，这个过程发生的水平很低。