Isolation, characterization, and transplantation of candidate stem cells

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造血谱系，并且在过去几十年中它们的广泛表征已经导致临床应用，包括骨髓移植用于影响造血区室的良性和恶性病症。 基于CD 34抗原表达的骨髓细胞分离富集原始区室，并且分离CD 34阳性群体的技术通常用于临床实践。 真正的造血干细胞的检测需要移植和再增殖，因此在人类细胞中查询这种潜力的研究是困难的。 因此，我们已经开发了具有稳健的人造血干细胞植入的异种移植模型，并且已经证明了所有细胞谱系的植入。 该试验将使我们能够在相关模型中首次在移植的人类细胞中测试珠蛋白载体。 最近，Goodell等描述了一种在致死辐射后仅基于用活体染料Hoechst 33342染色后的双波长流式细胞术分离能够重建造血的鼠骨髓细胞的方法。这些侧群细胞的分子基础最近归因于ABC转运蛋白ABCG 2的表达。侧群（SP）细胞高度富集造血重建能力，其中一个细胞能够在辐射受体小鼠中重建造血。 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parynchymal部分分离SP细胞，并在肝培养基中培养这些细胞。 肝SP细胞产生的细胞集落显示出富含颗粒的细胞质和致密的，通常为与肝细胞一致的双核。 这些肝细胞样细胞表达人肝细胞的标志物，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、沿着更特异性的标志物α-1-抗胰蛋白酶和人P450基因CYP 2B 6。 我们现在已经开发了一种在NOD/SCID小鼠中用亚致死剂量的四氯化碳治疗的体内救援模型，并且初步结果表明，与对照相比，SP细胞能够影响与成熟肝细胞类似的肝功能测试的正常化。这些SP细胞的进一步体内表征正在进行中。 此外，SP细胞已从成人胰腺中分离出来，微阵列分析表明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只取得了有限的成功，这些研究正在进行中，但它们在培养中的静止引起了β细胞是否可以在成年宿主中再生的问题。因此，我们开发了一种方法，用于评估β细胞再生出生后使用14 C测年的加速器质谱。 初步结果显示营业额有限。 此外，从接受DNA标记剂BrdU和IdU的个体获得样品并分析周转的证据。 这些样品还表明成体β细胞的更新有限。因此，我们又回到动物模型，以进一步询问I型糖尿病耐受诱导的潜力，因为这种耐受在许多疾病状态中是必要的，包括我们的主要焦点，异基因造血干细胞移植。 正在进行利用雷帕霉素的研究，我们已经证明雷帕霉素在动物模型和人类中实现了操作耐受性，以确定该药剂是否可以在小鼠中天然存在的I型死亡模型中导致抗原特异性耐受性。 这项工作将用于支持与该领域临床研究人员的合作研究。

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.