Isolation, characterization, and transplantation of stem

造血干细胞在出生后存在于骨髓中，为宿主的生命提供所有的造血系，在过去的几十年里，它们的广泛特征导致了临床应用，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的分离丰富了原始的隔室，而分离CD34阳性群体的技术在临床上是常用的。最近，Goodell et描述了一种完全基于双波长流式细胞术的小鼠骨髓细胞的分离方法，该细胞经活性染料Hoechst 33342染色后，能够在致死照射后重建造血。艾尔这些侧群细胞的分子基础最近被归因于ABC转运蛋白ABCG2的表达。侧群(SP)细胞具有高度的造血再繁殖能力，其中一种细胞能够在受照射的小鼠中重建造血。我们推测，这种表型在具有出生后再生能力的器官中是保守的。我们最近从人身体肝脏的非合并部分离出SP细胞，并将这些细胞培养在肝脏培养液中。肝SP细胞产生大量细胞集落，胞浆颗粒丰富，胞核致密，常为双核，与肝细胞一致。这些肝细胞样细胞表达人肝细胞标志物，包括HepPar、细胞角蛋白-8和人白蛋白。RT-PCR证实了肝细胞标志物的表达，包括白蛋白、细胞角蛋白-18，以及更特异的标志物-1抗胰蛋白酶和人类P450基因CYP2B6。我们现在已经建立了一个用亚致死剂量的四氯化碳治疗的NOD/SCID小鼠的体内救援模型，初步结果表明，与对照组相比，SP细胞能够影响与成熟肝细胞相似的肝功能测试的正常化。对这些SP细胞的进一步体内鉴定正在进行中。此外，已从成人胰腺分离出SP细胞，微阵列分析显示基因表达谱与骨髓来源的SP细胞相似。将胰腺SP细胞分化为成熟的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。最后，我们克隆了恒河猴ABCG2基因，以确定该转运蛋白在原始造血室强制表达的效果。我们的结果证实了恒河猴ABCG2的存在，确定了其在大型动物中赋予SP表型的重要性，表明其过度表达对体内分化没有不利影响，并暗示其过度表达作为基因治疗应用的体内选择策略1的潜在作用。

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. Finally, we have cloned the rhesus ABCG2 gene to determine the effects of forced expression of this transporter in the primitive hematopoietic compartment. Our results confirm the existence of rhesus-ABCG2, establish its importance in conferring the SP phenotype in large animals, suggest no detrimental effect of its overexpression upon differentiation in vivo, and imply a potential role for its overexpression as an in vivo selection strategy for gene therapy applications1.