Isolation, characterization, and transplantation of candidate stem cells

候选干细胞的分离、表征和移植

• 批准号: 8557973
• 负责人: John Tisdale
• 金额: $ 58.6万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 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/8557973
• 关键词: ABCG2 gene; ATP-Binding Cassette Transporters; Adult; Affect; Albumins; Allogenic; Animal Model; Antigens; Back; Benign; Beta Cell; Biological Assay; Bone Marrow; Bone Marrow Cells; Bone Marrow Transplantation; Bromodeoxyuridine; CD34 Antigens; CD34 gene; CYP2B6 gene; Carbon; Cell Lineage; Cell Nucleus; Cells; Chloride Ion; Chlorides; Clinical Investigator; Culture Media; Cultured Cells; Cytochrome P450; Cytokeratin 18; Cytokeratin 8; Cytoplasm; Cytoplasmic Granules; DNA; Disease; Dose; Dyes; Engraftment; Flow Cytometry; Gene Expression Profile; Globin; Hematopoiesis; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Hepatic; Hepatocyte; Human; Individual; Insulin-Dependent Diabetes Mellitus; Knowledge; Label; Lead; Life; Liver; Liver Function Tests; Malignant - descriptor; Methods; Microarray Analysis; Modeling; Molecular; Mus; NOD/SCID mouse; Natural regeneration; Organ; Pancreas; Phenotype; Population; Reverse Transcriptase Polymerase Chain Reaction; Sampling; Side; Sirolimus; Source; Speed; Staining method; Stains; Stem cells; Stress; Techniques; Testing; Time; Transplantation; Work; Xenograft Model; accelerator mass spectrometry; alpha 1-Antitrypsin; base; clinical application; clinical practice; human disease; in vivo; irradiation; meetings; reconstitution; repaired; success; vector

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. Assays for true hematopoietic stem cells require transplantation and repopulation, and thus studies querying such potential among human cells are difficult. We have thus developed a xenograft model with robust human hematopoietic stem cell engraftment, and have demonstrated engraftment of all cell lineages. This assay will allow us to test globin vectors for the first time among engrafted human cells in a relevant model. 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, alpha-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. Further, samples from individuals who received the DNA labeling agents BrdU and IdU were obtained and analyzed for evidence of turnover. These samples also suggest limited turnover of adult beta cells. We have thus turned back to animal models to further query the potential for tolerance induction in Type I diabetes mellitus, as this tolerance will be necessary in a number of disease states, including our main focus, allogeneic hematopoietic stem cell transplantation. Studies are ongoing utilizing rapamycin, an agent we have demonstrated to achieve operational tolerance in animal models and humans, to determine if this agent can lead to antigen specific tolerance in a naturally occurring model of Type I dieabetes in the mouse. This work will be used to support collaborative studies with clinical investigators in the field.

造血干细胞在出生后存在于骨髓中，为宿主的生命提供了所有的造血谱系，在过去的几十年里，它们的广泛特性导致了临床应用，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丰富了原始隔室，分离CD34阳性群体的技术在临床实践中常用。真正的造血干细胞的检测需要移植和再生，因此在人类细胞中质疑这种潜力的研究是困难的。因此，我们开发了一种具有强大的人类造血干细胞移植的异种移植模型，并证明了所有细胞系的移植。该试验将使我们能够首次在相关模型中移植的人类细胞中测试珠蛋白载体。