Organ specificity of c-kit+ lymphoid precursors in the liver, thymus, and bone marrow

c-kit+Lin- cells are present in various immune organs, including the liver, thymus, and bone marrow, where lymphoid, myeloid, or erythroid cells are generated. To compare their properties as lymphoid precursors, c-kit+Lin- cells purified from various organs of B6.Ly5.1 mice were injected into 6.5 Gy-irradiated B6.Ly5.2 mice. Depending on the source of the c-kit cells, the degree of entrance and expansion of lymphoid cells differed in the liver and thymus of recipient mice. c-kit+ cells isolated from the bone marrow entered and expanded prominently in both the liver and thymus, whereas c-kit+ cells from the thymus did not do so at all. On the other hand, c-kit+ cells isolated from the liver and spleen showed an intermediate pattern, namely, they took a long time to enter and expand in the liver and thymus of recipient mice. All of these c-kit+ cells had the potential to give rise to lymphoid cells, which were specific to the liver and thymus, respectively. We previously showed that progenitor cells for extrathymic T cells in the liver and those for conventional T cells in the thymus are not always supplied by the bone marrow, as shown by experiments using parabiosis. Taken together with those previous data, the present results suggest that c-kit+Lin- cells isolated from various immune organs have organ specific properties.     

骨髓干细胞在大鼠肝再生环境中的分化

研究大鼠骨髓干细胞在部分肝切除后肝再生环境中的分化。从部分肝切除模型大鼠的胫骨中提取骨髓细胞,应用流式细胞仪富集骨髓干细胞,以PKH26-GL体外标记后通过门静脉进行自体移植,2周后行白蛋白和角蛋白8免疫组化检查。结果肝板肝细胞间PKH26-GL标记骨髓干细胞表达白蛋白、角蛋白8。提示骨髓干细胞在部分肝切除后肝再生环境中能分化为肝细胞,骨髓干细胞可能参与部分肝切除后的肝再生过程。     

骨髓间充质干细胞移植在肝再生中的应用

随着当今医学的发展,干细胞的应用逐渐涉及到各个领域,并发挥着无限的潜能。干细胞以其自我更新及多向分化的能力可以对多种疾病进行替代治疗。肝病终末期肝细胞数量减少,肝功能衰竭,通过干细胞的替代治疗,肝功能能够得以改善,肝纤维化得到缓解甚至逆转。干细胞治疗成为有望替代肝移植的新技术。此文就干细胞在肝病治疗中的潜能作一综述,包括干细胞的来源、治疗机制、治疗途径,并对其临床应用进行探讨。     

骨髓干细胞移植治疗急性肝衰竭的实验研究

目的探讨骨髓干细胞移植治疗实验性肝衰竭大鼠的效率及可行性。方法以D-氨基半乳糖及内毒素制备雌性大鼠肝衰竭模型,然后动员和富集雄性大鼠骨髓干细胞,并经门静脉移植至雌性大鼠肝脏;移植后观察受体雌鼠临床表现、生化改变、肝脏病变及肝组织Y染色体阳性率。结果受体肝衰竭雌鼠在细胞移植后,精神、食欲逐渐好转,生化异常及肝脏病变得以恢复,肝脏可检出Y染色体阳性的供体源性肝细胞;未接受骨髓干细胞移植的对照组雌鼠则全部死亡。结论骨髓干细胞移植对实验性肝衰竭大鼠具有明确治疗作用。     

Erythropoietin response in anaemic patients with multiple myeloma and other lymphoid malignancies infiltrating the bone marrow.

Immunoreactive erythropoietin levels were measured in 42 patients with lymphoid malignancies with anaemia and bone marrow involvement. Results were compared to a control group of 16 patients suffering from anaemia due to other causes. Significant inverse correlations between serum erythropoietin level and haemoglobin concentration were shown for the patients with lymphoid malignancies and also for the control subjects. Overall, the erythropoietin levels of patients with lymphoid malignancies with bone marrow infiltration and with normal renal function did not differ significantly from erythropoietin levels of the anaemic controls. We conclude that anaemia in patients with lymphoproliferative disorders with bone marrow infiltration and normal renal function is caused primarily by a diminished/inadequate response to erythropoietin at the level of the target cell.     

Exclusive expression of G-CSF receptor on myeloid progenitors in bone marrow CD34+ cells

Although granulocyte colony-stimulating factor (G-CSF) has been reported to act on cells of neutrophilic lineage, the administration of G-CSF to induce the mobilization of various haematopoietic progenitors into the circulation. We analysed the expression of receptors for G-CSF (G-CSFR) on human bone marrow and G-CSF-mobilized peripheral blood CD34+ cells, and examined the proliferation and differentiation capabilities of sorted CD34+G-CSFR+ and CD34+G-CSFR- cells using methylcellulose clonal culture. Flow cytometric analysis showed that G-CSFR was expressed on 14.9 +/- 4.9% of bone marrow CD34+ cells, most of which were included in CD34+CD33+ and CD34+CD38+ cell fractions. In clonal cultures, CD34+G-CSFR+ cells produced only myeloid colonies, whereas CD34+G-CSFR- cells produced erythroid bursts, megakaryocyte and multilineage colonies. When incubated with the cytokine cocktail for 5 d, CD34+G-CSFR- cells generated CD34+G-CSFR+ myeloid progenitors. In G-CSF-mobilized peripheral blood, CD34+ cells contained 10.8 +/- 5.8% of G-CSFR+ cells, most of which were also myeloid progenitors, although CD34+G-CSFR- cells contained a substantial number of myeloid progenitors. These results indicated that the expression of G-CSFR on CD34+ cells is restricted to myeloid progenitors, suggesting that the specific activity of G-CSF on myelopoiesis depends on the exclusive expression of its receptor on myeloid progenitors, and that the mobilization of various haematopoietic progenitors is not a direct effect of G-CSF in humans.     

自体骨髓干细胞移植治疗终末期肝病56例临床观察

目的：探讨自体骨髓干细胞移植对终末期肝病患者的治疗作用。方法：56例终末期肝病患者无菌采集骨髓,负收集法分离纯化骨髓干细胞,经肝固有动脉注入肝脏。术后观察肝脏储备功能和合成功能,同时观察患者症状体征和不良反应。结果：患者骨髓采集量为120～180ml,分离细胞数为5．8×10^7-9．6×10^8个,与术前比较,术后1周91．6％的患者食欲改善,87．5％的患者体力明显改善;术后12周吲哚氰绿血浆清除率（ICGK）升高,吲哚氰绿15min滞留率（ICGRl5）下降,与术前比较差异有显著性意义（P〈0．05）;血清血蛋白（Alb）在术后4周开始升高,12周达到最高,与治疗前比差异有显著性意义（P〈0．05）;胆碱酯酶（CHE）、凝血酶原时间（PT）移植术后持续改善,12周时较术前增加最明显,差异有显著性意义（P〈0．05）;18例并发腹水者,有11例患者腹水消失,有5例腹水减轻但没有消退。结论：自体骨髓干细胞移植技术治疗终末期肝病,患者肝脏储备功能、合成功能改善明显,术后无不良反应,安全可行。     

骨髓间充质干细胞移植治疗大鼠急性肝功能衰竭

目的探讨骨髓间充质干细胞（BMSCs）移植对急性肝功能衰竭的治疗作用。方法大鼠90%肝脏切除制备急性肝功能衰竭模型,实验组大鼠肝内移植2×106个BMSCs,对照组仅注射生理盐水。观察大鼠的生存情况,RT-PCR检测BMSCs在肝脏的植入,血清检测肝功能确认BMSCs移植对肝脏修复的作用。结果移植BM-SCs的实验组大鼠存活80%,对照组大鼠仅存活20%。RT-PCR显示,BMSCs移植后定植于大鼠肝脏内。肝功能检测显示,实验组大鼠的血清丙氨酸氨基转移酶水平、天冬氨酸氨基转移酶水平显著降低,血清白蛋白水平显著升高。结论 BMSCs移植可以显著提高急性功能衰竭大鼠的生存率,促进其肝功能的恢复,对急性肝功能衰竭的治疗具有积极作用。     

Haemopoietic defect and decreased expansion potential of bone marrow autografts from patients with acute myeloid leukaemia in first remission

Autologous bone marrow (BM) transplantation for acute myeloid leukaemia (AML) in complete remission (CR) is frequently followed by a slow haemopoietic recovery. We assessed the haemopoietic capacity of purified BM stem cell (CD34⁺DR^?) and progenitor cell (CD34⁺DR⁺) populations from patients with AML in CR, and compared these data with those of normal BM. The feasibility of ex vivo expansion in stroma-conditioned medium supplemented with cytokines was also investigated. The number of CFU-GM produced by initial patient CD34⁺DR^? cells was decreased compared to normal, whereas these values were similar to normal for CD34⁺DR⁺ cells. BFU-E, HPP-CFC and LTC-IC were reduced for both patient CD34⁺DR^? and CD34⁺DR⁺ subpopulations. In contrast to normal, the patient CD34⁺DR^? fraction was not enriched in LTC-IC. CFU-GM expansion from patient CD34⁺DR^? cells was poor and decreased after 14 d of culture. No HPP-CFC expansion could be observed for patient cells. LTC-IC were below the level of detection after 14–21 d of expansion culture of CD34⁺DR^? patient cells, whereas they were variably maintained or expanded for normal cells. After expansion culture, cytogenetic and/or FISH analyses did not reveal the anomalies present at diagnosis, regardless of the cell subpopulation analysed. In conclusion, BM cells of patients with AML in CR show a profound defect at the level of a stem cell enriched population. No meaningful ex vivo expansion could be obtained in culture conditions allowing for a significant expansion from a normal stem cell population.     

Differentiation and expansion of endothelial cells from human bone marrow CD133+ cells

We report a method of purifying, characterizing and expanding endothelial cells (ECs) derived from CD133(+) bone marrow cells, a subset of CD34(+) haematopoietic progenitors. Isolated using immunomagnetic sorting (mean purity 90 +/- 5%), the CD133(+) bone marrow cells were grown on fibronectin-coated flasks in M199 medium supplemented with fetal bovine serum (FBS), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and insulin growth factor (IGF-1). The CD133(+) fraction contained 95 +/- 4% CD34(+) cells, 3 +/- 2% cells expressing VEGF receptor (VEGFR-2/KDR), but did not express von Willebrand factor (VWF), VE-cadherin, P1H12 or TE-7. After 3 weeks of culture, the cells formed a monolayer with a typical EC morphology and expanded 11 +/- 5 times. The cells were further purified using Ulex europaeus agglutinin-1 (UEA-1)-fluorescein isothiocyanate (FITC) and anti-FITC microbeads, and expanded with VEGF for a further 3 weeks. All of the cells were CD45(-) and CD14(-), and expressed several endothelial markers (UEA-1, VWF, P1H12, CD105, E-selectin, VCAM-1 and VE-cadherin) and typical Weibel-Palade bodies. They had a high proliferative potential (up to a 2400-fold increase in cell number after 3 weeks of culture) and the capacity to modulate cell surface antigens upon stimulation with inflammatory cytokines. Purified ECs were also co-cultivated with CD34(+) cells, in parallel with a purified fibroblastic cell monolayer. CD34(+) cells (10 x 10(5)) gave rise to 17,951 +/- 2422 CFU-GM colonies when grown on endothelial cells, and to 12,928 +/- 4415 CFU-GM colonies on fibroblast monolayers. The ECs also supported erythroid blast-forming unit (BFU-E) colonies better. These results suggest that bone marrow CD133(+) progenitor cells can give rise to highly purified ECs, which have a high proliferative capacity, can be activated by inflammatory cytokines and are superior to fibroblasts in supporting haematopoiesis. Our data support the hypothesis that endothelial cell progenitors are present in adult bone marrow and may contribute to neo-angiogenesis.     

Human bone marrow adipocytes support complete myeloid and lymphoid differentiation from human CD34 cells

In humans, the role of bone marrow (BM) adipocytes in supporting haematopoiesis has been questioned. A co-culture system of CD34(+) cells seeded onto either BM undifferentiated mesenchymal stem cells or differentiated adipocytes showed that BM adipocytes did not support the maintenance of immature progenitors but enabled their complete differentiation along the myeloid and lymphoid pathways. These properties appear to be opposite to those of osteoblasts, although both cell types share a common mesenchymal progenitor. These results suggest that stromal cells play a variety of roles in the haematopoietic microenvironment, which could be significant in situations such as osteoporosis or ageing.     

自体骨髓干细胞移植治疗肝衰竭的初步观察

目的观察自体骨髓干细胞移植治疗肝衰竭的初步疗效。方法 12例经常规内科综合治疗无明显效果的肝衰竭患者。在无菌条件下采集骨髓100～200ml,通过密度梯度离心分离纯化出骨髓干细胞,通过介入的方法经肝动脉注射入肝内,静脉输注肝细胞生长素1～2周。观察移植后1、2、3和6月患者症状、体征、肝脏生化和凝血酶原时间等的变化。结果 9例肝衰竭患者的症状、体征改善,白蛋白升高、胆红素降低,肝脏功能改善,所有患者未见明显副作用。结论自体骨髓干细胞移植治疗肝衰竭安全有效。     

罗曼鹤鸡骨髓间充质干细胞的分离培养和鉴定

目的建立鸡骨髓间充质干细胞（BMSCs）的分离培养和鉴定体系。方法从1～14天龄罗曼鹤鸡骨髓中分离骨髓细胞,差速贴壁法纯化、扩增BMSCs。倒置显微镜下观察细胞形态特征,MTY法绘制生长曲线,流式细胞仪检测细胞标志物,并分别进行成骨、成脂诱导分化能力检测。结果分离培养的细胞呈成纤维细胞样或长梭形,生长状态良好;CD29阳性表达率为92．10％,CD34阳性表达率仅为0．80％;经成骨诱导分化,出现明显的钙化结节,茜素红染色阳性;经成脂诱导分化,油红O染色阳性,细胞内出现明显的脂质小滴。结论建立了操作简单、高效的鸡BMSCs分离培养和鉴定体系,为鸡BMSCs的进一步研究和应用提供了良好的基础。     

Prolonged ex vivo culture of human bone marrow mesenchymal stem cells influences their supportive activity toward NOD/SCID-repopulating cells and committed progenitor cells of B lymphoid and myeloid lineages

Background

Bone marrow mesenchymal stem cells support proliferation and differentiation of hematopoietic progenitor cells in vitro. Since these cells constitute a rare subset of bone marrow cells, mesenchymal stem cell preparations for clinical purposes require a preparative step of ex vivo multiplication. The aim of our study was to analyze the influence of culture duration on mesenchymal stem cell supportive activity.

Design and Methods

Mesenchymal stem cells were expanded for up to ten passages. These cells and CD34+ cells were seeded in cytokine-free co-cultures after which the phenotype, clonogenic capacity and in vivo repopulating activity of harvested hematopoietic cells were assessed.

Results

Early passage mesenchymal stem cells supported hematopoietic progenitor cell expansion and differentiation toward both B lymphoid and myeloid lineages. Late passage mesenchymal stem cells did not support hematopoietic progenitor cell and myeloid cell outgrowth but maintained B-cell supportive ability. In vitro maintenance of NOD/SCID mouse repopulating cells cultured for 1 week in contact with mesenchymal stem cells was effective until the fourth passage of the mesenchymal cells and declined thereafter. The levels of engraftment of CD34⁺ cells in NOD/SCID mice was higher when these cells were co-injected with early passage mesenchymal stem cells; however mesenchymal cells expanded beyond nine passages were ineffective in promoting CD34⁺ cell engraftment. Non-contact cultures indicated that mesenchymal stem cell supportive activity involved diffusible factors. Among these, interleukins 6 and 8 contributed to the supportive activity of early passage mesenchymal stem cells but not to those of late passage cells. The phenotype, as well as fat, bone and cartilage differentiation capacity, of mesenchymal stem cells did not change during their culture.

Conclusions

Extended culture of mesenchymal stem cells alters the ability of these cells to support hematopoietic progenitor cells without causing concomitant changes in their phenotype or differentiation capacity.     

Different expression of adhesion molecules on myeloid and B-lymphoid CD34+ progenitors in normal bone marrow

Abstract: The expression of adhesion molecules was studied on B lymphoid and myeloid CD34⁺ precursors in normal bone marrow. Bone marrow aspirates were labelled in a double fluorescence procedure with the CD34 monoclonal antibody 43A1 and with antibodies directed against maturation and differentiation antigens and adhesion molecules. Three clusters of CD34⁺ cells could be distinguished by their light scatter characteristics in flow cytometry. The population with the lowest forward scatter contained B-lymphoid precursors while the two others showed phenotypic characteristics of, respectively, early and late myeloid precursors. Nearly all CD34⁺ cells in the 3 subpopulations expressed VLA-4, VLA-5, LFA-3 and H-CAM. B-lymphoid progenitors showed a higher density of VLA-4 and VLA-5 than the myeloid progenitors. Myeloid precursors, and particularly the late subset, expressed more HCAM than the B-lymphoid progenitors. The majority of the CD34⁺ cells also expressed LFA-1 and L-selectin. Higher numbers of positive cells were found in the myeloid subset. The early myeloid subset showed the highest positivity for L-selectin. We conclude that B lymphoid and early and late myeloid CD34⁺ precursors in normal bone marrow show a different profile of adhesion molecules. These profiles could reflect a higher tendency of the myeloid CD34⁺ precursors to circulate.     

Correlation of c-kit expression and cell cycle regulation by transforming growth factor-beta in CD34+ CD38- human bone marrow cells

To investigate the relationship between c-kit expression and cell cycle regulation by endogenous transforming growth factor-beta (TGF-beta) in human bone marrow hematopoietic progenitor cells, CD34+ CD38- c-kit(low/-) and CD34+ CD38- c-kit(high) populations were cultured in stem cell factor, thrombopoietin, interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor, granulocyte/macrophage colony-stimulating factor and anti-TGF-beta, and analyzed for cell cycle status. Arrest in G0/G1 was most prominent in the precultured CD34+ CD38- c-kit(low/-) subset (95.62 +/- 4.15%). While postcultured CD34+ CD38- c-kit(high) cells initiated from CD34+ CD38- c-kit(high) cells entered cell cycle within 36 hr, postcultured CD34+ CD38- c-kit(low/-) cells initiated from CD34+ CD38- c-kit(low/-) cells remained dormant until 36 hr and entered cell cycle within 90 hr. Anti-TGF-beta increased the percentage of S/G2M phase postcultured CD34+ CD38- c-kit(high) cells (from 19.08 +/- 11.95 to 47.04 +/- 2.93%), but no significant change was observed in postcultured CD34+ CD38- c-kit(low/-) cells. These results suggest that endogenous TGF-beta plays an important role in the cell cycle arrest of c-kit(high) but not c-kit(low/-) cells in CD34+ CD38- cells, which proliferate without undergoing differentiation. The different regulatory mechanism of cell cycle entry of the CD34+ CD38- c-kit(high) and CD34+ CD38- c-kit(low/-) subsets might be the result of differences in their sensitivity to endogenous TGF-beta.     

骨髓干细胞治疗慢性肝衰竭的研究进展

肝移植是治疗慢性肝衰竭(chronic liver failure,CLF)的有效手段,但因各种原因,其临床应用逐渐受到限制,探索新的治疗手段迫在眉睫.1996年Alison等诱导人骨髓干细胞(bone marrow stem cells,BMSCs)分化肝细胞获得成功,推动了BMSCs治疗CLF研究的快速发展.     

Autologous bone marrow cell infusion therapy for liver cirrhosis

The plasticity of bone marrow cells (BMC) has been confirmed by autopsy results of female recipients of BMC from male donors. To establish new clinical therapies for patients with liver cirrhosis using autologous BMC, we developed a new in vivo murine model using green fluorescent protein (GFP) and repeated carbon tetrachloride (CCl₄) injection. We found that BMC infused through the tail vein, efficiently repopulated cirrhotic liver tissue and, under the influence of persistent liver damage induced by carbon tetrachloride, differentiated into albumin-producing hepatocytes. Moreover, such BMC infusions into mice with cirrhosis improved liver function and reduced mortality. The latter observation correlated with the strong expression of matrix metalloproteinases (MMP), particularly MMP-9, and reduced hepatic fibrosis. The results from the 'GFP/CCl₄ model' showed that cell therapy using autologous BMC has the potential to become an effective treatment for patients with liver failure due to advanced liver cirrhosis. This review summarizes previous findings plus these recent experimental results, as well as recent clinical trials of BMC transfusion into patients with end-stage chronic liver disease.     

白血病干细胞和骨髓基质细胞共培养的方法学探讨

目的 探讨白血病干细胞和骨髓基质细胞共培养的可行方法及扩增白血病干细胞的特点.方法 体外用两种方法共培养急性髓系白血病（AML）患者的骨髓单个核细胞（MNCs）：一种方法是将MNCs中的贴壁基质细胞单独培养,第2代或第3代基质细胞用丝裂霉素处理后做饲养层,再同悬浮造血细胞共培养;另一种方法是将MNCs持续培养即原代共培养.观察MNCs形态学特征、长期存活情况及增殖特性,用流式细胞仪检测分析细胞表面标志.结果 两种方法共培养的细胞部分可以长期存活,在共培养中有卵石样区域细胞及悬浮细胞团的形成.原代共培养的细胞所形成的卵石样区域数目多,排列规律.结论 两种方法都可以起到扩增白血病干细胞的作用.同经过丝裂霉素处理之后的饲养层相比,没有经过处理的骨髓基质细胞在共培养时能够更有效地扩增白血病干细胞.