骨髓干细胞转分化为肝细胞的研究进展

目前，有关骨髓干细胞的转分化现象已成为国际上干细胞的研究热点之一。1997年，Eglitis等发现供体来源的骨髓干细胞能分化为神经胶质细胞，表明骨髓干细胞在一定条件下可以跨胚层分化成其他类型的细胞，这种现象被称为转分化／横向分化（transdifferentiation）。1999年，Petersen等首次报道骨髓干细胞可以转分化为肝脏细胞后，人们希望骨髓干细胞能成为治疗急、慢性肝衰竭的一个新方法。但是目前对于骨髓干细胞转化为肝细胞的研究仍存在许多谜团和争议。因此，本文着重对近年来骨髓来源的肝细胞的研究进行综述。     

Spontaneous immunoglobulin secreting cells in idiopathic usual interstitial pneumonia

Abundant evidence suggests that activation of B lymphocytes and formation of immune complexes play important roles in the pathogenesis of idiopathic pulmonary fibrosis. We investigated the level of B lymphocyte activation in patients with idiopathic UIP (n = 10, all were biopsy proven cases), for the purpose of evaluating its role. In order to determine the level of B lymphocyte activation, the number of spontaneous immunoglobulin secreting cells in blood was counted by using reverse hemolytic plaque assay. The number of IgA and IgG secreting cells significantly increased in patients with idiopathic UIP when compared with those in healthy controls (n = 8). The number of IgG secreting cells also significantly increased in patients with IP-CVD, but no significant increase in the number of Ig secreting cells could be detected in patients with pulmonary emphysema, diffuse panbronchiolitis or sarcoidosis. The study of cell surface markers revealed significant increase of CD21 (B2 and OKB7) positive cells in patients with idiopathic UIP. CD21 positive cells were immature B lymphocytes. These results suggest that increase of resting B lymphocytes and immunoglobulin secreting cells occurred and that the differentiation of B lymphocytes may be accelerated in idiopathic UIP. Furthermore, there is the possibility that soluble factors produced by T lymphocytes influenced the function of B lymphocytes in our experiment.     

Exact relevance of bone marrow cells in the healing process after myocardial infarction: analysis with a murine model of bone marrow cell transplantation

BACKGROUND: Cellular cardiomyoplasty has created new possibilities in cardiac regeneration. Several cell types can be used in the procedure, such as skeletal myoblasts and bone marrow cells. Recent publications have suggested that bone marrow cells may be excellent candidates due to their pluripotency, but their actual role in cardiac regeneration is unknown. OBJECTIVE: To evaluate the exact physiological role of bone marrow cells in the healing process after myocardial infarction. METHODS: A mouse bone marrow cell transplantation model was used in which transplanted cells were easily detectable by immunohistochemistry. Chimeric mice were subjected to myocardial infarction by ligation of the left descending coronary artery. After one month, the mice were sacrificed and the scars were analyzed. RESULTS: Transplanted bone marrow cells were detected in the scars and these cells seemed able to transdifferentiate into endothelial cells, but no transdifferentiation into cardiomyocytes occurred. This mechanism of regeneration was dismissed because only 2% of the vessels in the scars were positive for transplanted cells. CONCLUSIONS: Bone marrow cells might be involved in myocardial healing, but this physiological mechanism is insufficient to allow correct regeneration.     

Granulocyte-colony stimulating factor increases donor mesenchymal stem cells in bone marrow and their mobilization into peripheral circulation but does not repair dystrophic heart after bone marrow transplantation

BACKGROUND: Hereditary disordered cardiac muscle could be replaced with intact cardiomyocytes derived from genetically intact bone marrow (BM)-derived stem cells. METHODS AND RESULTS: Cardiomyopathic mice with targeted mutation of delta-sarcoglycan gene underwent intra-BM-BM transplantation (IBM-BMT) from transgenic mice expressing green fluorescence protein. The host BM and the peripheral blood were completely reconstituted by donor-derived hematopoietic cells by IBM-BMT. Treatment with granulocyte-colony stimulating factor (G-CSF) markedly increased donor-derived mesenchymal stem cells (MSC) in the BM and their mobilization into the peripheral blood after IBM-BMT. Treatment with isoproterenol (iso) for 7 days caused myocardial damage and left ventricular (LV) dysfunction in the cardiomyopathic mice. Co-treatment with iso and G-CSF increased donor BM cell recruitment to the heart and temporarily improved LV function in the cardiomyopathic mice with or without IBM-BMT. However, the cardiac muscle was not replaced with donor BM-derived cardiomyocytes in the cardiomyopathic mice with or without IBM-BMT, and this was associated with no improvement of LV function of mice aged 20 weeks. CONCLUSIONS: These results suggest that G-CSF enhances engraftment of donor MSC in the BM and their mobilization into the peripheral circulation after IBM-BMT but MSC recruited to the heart do not differentiate into cardiomyocytes and do not repair the dystrophic heart.     

Unique patterns of surface receptors, cytokine secretion, and immune functions distinguish T cells in the bone marrow from those in the periphery: impact on allogeneic bone marrow transplantation

The "conventional" NK1.1(-) T cells from mouse blood and marrow were compared with regard to surface receptors, cytokine secretion, and function. Most blood NK1.1(-) CD4(+) and CD8(+) T cells expressed the naive CD44(int/lo)CD62L(hi)CD45RB(hi) T-cell phenotype typical of those in the peripheral lymphoid tissues. In contrast, most marrow NK1.1(-) CD4(+) and CD8(+) T cells expressed an unusual CD44(hi)CD62L(hi)CD45RB(hi) phenotype. The blood NK1.1(-) CD4(+) T cells had a naive T-helper cytokine profile and a potent capacity to induce lethal graft versus host (GVH) disease in a C57BL/6 donor to a BALB/c host bone marrow transplantation model. In contrast, the marrow NK1.1(-) CD4(+) T cells had a Th0 cytokine profile and failed to induce lethal GVH disease, even at 20-fold higher numbers than those from the blood. NK1.1(-) CD8(+) T cells from the blood but not the marrow induced lethal GVH disease. Nevertheless, the marrow NK1.1(-) CD8(+) T cells induced potent antitumor activity that was augmented by marrow NK1.1(-) CD4(+) T cells and facilitated hematopoietic progenitor engraftment. The inability of marrow CD4(+) and CD8(+) T cells to induce GVH disease was associated with their inability to expand in the blood and gut of allogeneic recipients. Because neither the purified marrow CD4(+) or CD8(+) T cells induced GVH disease, their unique features are desirable for inclusion in allogeneic bone marrow or hematopoietic progenitor transplants.     

Activation of cardiomyocytes depending on their proximity to human bone marrow stem cells

Our study aimed to elucidate whether bone marrow stem cell (BMC) treatment might result in a cellular response in cardiomyocytes IN VITRO. Subconfluent neonatal rat cardiomyocyte cultures were cocultured for three days with Vybrant CM-DiI labeled BMC from human sternal bone marrow and underwent immunohistological staining for the proto-oncogene c-Myc and the cell cycle proteins CDK2, CDK4 and ATF-3. β-adrenoceptor density was analyzed using [125I]-iodocyanopindolol (ICYP) histoautoradiography. Quantitative analysis of immunohistochemical images revealed significantly increased expression and upregulation of c-Myc, and its downstream targets ATF-3, CDK2 and CDK4 in neighboring cardiomyocytes to BMC, depending on their distance to the BMC compared to cardiomyocytes far from the BMC. Histoautoradiography revealed a significantly higher β-adrenoceptor density in cardiomyocytes in the immediate vicinity to the BMC. With increasing distance to the BMC, β-adrenoceptor density in cardiomyocytes declined. Thus, a small number of BMC can affect a larger number of cardiomyocytes by activating an intracellular signaling cascade and enhancing β-adrenoceptor density.     

Depletion of T cells from bone marrow for allogeneic transplantation: method for treatment of bone marrow in bulk

T lymphocytes were depleted from donor marrow for 23 patients undergoing allogeneic bone marrow transplantation using an anti-T-cell antibody, CT-2, and complement. The methodology is described in detail for in vitro depletion of large quantities of bone marrow. The extent of T-lymphocyte depletion using various T-cell markers, the percent of marrow lost in the processing and quantity of antibody, and complement needed are presented. These techniques for in vitro T-lymphocyte depletion were reproducible and did result in an average final yield of 47% of the harvested donor marrow.     

Low level of gene transfer to and engraftment of murine bone marrow cells from long-term bone marrow cultures

OBJECTIVE: We wanted to determine whether the long-term bone marrow culture (LTBMC) transduction system would lead to efficient gene transfer and engraftment of murine repopulating hematopoietic stem cells (HSC), particularly in nonablated recipients. MATERIALS AND METHODS: Congenic mouse strains expressing Ly 5.1 or Ly 5.2 and the GP+E86 cell line producing the MGirL22Y vector carrying the gene for enhanced GFP were used. Murine LTBMCs were established and demi-depopulated on days 7 and 14 with addition of vector supernatant on days 8 and 15. RESULTS: Cell recovery on day 21 was 21.3%+/-3.8% of input cells and CFU-C recovery was 9.7+/-3.4% as compared with CFU-C of input cells. In vitro transduction efficiency determined by CFU-C expressing GFP was 22.2%+/-1.6%. In irradiated (950 cGy) mice transplanted with 2x10(6) LTBMC cells, 94% of nucleated cells in the blood at week 16 were of donor origin. However, GFP was only detected at low level in a few animals at week 4 and not later. Analysis of bone marrow from these mice at week 20 did not show any GFP expression and semiquantitative PCR revealed a transgene level of <1%. When 3.5-20.8x10(6) LTBMC cells (corresponding to 20-100x10(6) fresh cells) were transplanted to nonablated recipients, no engraftment or GFP expression were detected. Competitive repopulation experiments showed that the long-term repopulation ability (LTRA) of the LTMC cells was only 7% of fresh cells. CONCLUSION: These results indicate that LTBMC transduction of murine cells leads to low-level transduction of progenitors, no gene transfer to repopulating stem cells, and reduction in LTRA in ablated and nonablated recipients.     

内皮素-1对大鼠骨髓内皮前体细胞增殖、细胞周期及NO分泌功能的影响

目的探讨内皮素-1（ET-1）对大鼠骨髓内皮前体细胞（EPC）增殖、细胞周期及一氧化氮（NO）分泌功能的影响。方法采用密度梯度离心法获取大鼠骨髓单核细胞,在M199培养液中培养扩增EPC并进行鉴定。不同浓度ET-1（A组:0mol/L、B组:10-9mol/L、C组:10-8mol/L、D组:10-7mol/L、E组:10-6mol/L）作用于EPC,MTT法检测ET-1对EPC增殖能力的影响,流式细胞仪检测ET-1对EPC细胞周期的影响,硝酸还原酶法检测细胞培养液中NO含量的变化,观察ET-1对EPC的NO分泌功能的影响。结果与对照组（A组,0.405±0.017）相比,ET-1浓度C组（0.434±0.016）、D组（0.463±0.016）、E组（0.473±0.015）EPC增殖能力显著增高（n=8,P<0.01）。EPC的G0/G1期细胞百分数C组（57.28±3.65）%、D组（44.99±3.19）%、E组（40.29±3.74）%较对照组（70.55±1.37）%明显降低（n=5,P<0.01）,EPC的S期细胞百分数C组（26.75±2.87）%、D组（32.79±2.41）%、E组（35.74±2.94）%较对照组（20.04±1.64）%明显升高（n=5,P<0.01）,EPC的G2/M期细胞百分数C组（15.96±1.71）%、D组（22.22±2.22）%、E组（23.64±2.86）%较对照组（9.41±0.81）%明显升高（n=5,P<0.01）。EPC培养液中NO含量（μmol/L）ET-1浓度C组（11.70±1.80）、D组（15.69±1.86）、E组（16.89±1.55）较对照组（7.45±2.41）明显增加（n=8,P<0.01）。B组检测结果与对照组相比,各项指标均无统计学差异。结论ET-1能够促进EPC的增殖,促进细胞向S期和G2期的转化,并提高细胞NO分泌功能,可能参与缺血性疾病的血管再生过程。     

Differences in sulfation patterns of heparan sulfate derived from human bone marrow and umbilical vein endothelial cells.

OBJECTIVE: Heparan sulfates (HS), the polysaccharide side chains of HS proteoglycans, differ in structure and composition of sulfated domains among various tissue types, resulting in selective protein binding. HS proteoglycans on bone marrow endothelial cells (BMEC) could contribute to tissue specificity of the bone marrow endothelium and play a role in the presentation of chemokines such as stromal cell-derived factor-1 (SDF-1) and adhesion of hematopoietic progenitor cells after stem cell transplantations. We characterized differences in HS structure and SDF-1 binding between BMEC and human umbilical vein endothelial cells (HUVEC). MATERIALS AND METHODS: Expression of HS proteoglycans on human bone marrow microvessels was investigated by immunohistochemical staining. Comparison of three human BMEC cell lines with HUVEC and an HUVEC cell line was studied by flow cytometry using antibodies against different epitopes of the HS polysaccharide chain. HS proteoglycans were biochemically characterized after isolation from metabolically labeled cultures of the BMEC cell line 4LHBMEC and HUVEC. Binding of radiolabeled SDF-1 to 4LHBMEC and HUVEC and competition with heparins were investigated. RESULTS: Bone marrow microvessels constitutively expressed HS proteoglycans. Flow cytometric experiments showed differences in HS chain composition between BMEC and HUVEC. Biochemical characterization revealed more O-sulfation of the N-sulfated domains present in cell-associated HS glycosaminoglycans in 4LHBMEC compared to HUVEC. Binding experiments showed that 4LHBMEC bound more 125[I]-SDF-1 per cell than HUVEC. This could be inhibited largely by heparin and O-sulfated heparin and to a lesser extent by N-sulfated heparin. CONCLUSIONS: Cellular HS from BMEC differs in composition from HUVEC. We postulate that the presence of highly sulfated domains in the HS chains from BMEC contributes to tissue specificity of bone marrow endothelium in which HS may be involved in SDF-1 presentation and adhesion of hematopoietic progenitor cells.     

Role of hepatocyte growth factor in the development of dendritic cells from CD34+ bone marrow cells

BACKGROUND AND OBJECTIVE: Hepatocyte growth factor (HGF) is known to augment the effects of stem cell factor, interleukin-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoetin, and granulocyte colony-stimulating factor, all of which are involved in hematopoiesis. HGF is also known to have a role in immune responses. The aim of this study was to investigate whether HGF is involved in the development of dendritic cells (DC) from CD34+ bone marrow cells. DESIGN AND METHODS: CD34+ cells obtained from three healthy donors were incubated in various combinations of HGF, GM-CSF, and tumor necrosis factor (TNF) for 12 days. Developing cell populations were analyzed for surface markers, morphology and functional capacities by flow cytometry, light microscopy and mixed lymphocyte reaction, respectively. RESULTS: Incubation with HGF alone generated greater number of dendritic cells from CD34+ bone marrow cells than incubation with GM-CSF, or a combination of GM-CSF with TNF. HGF was also found to potentiate the effect of GM-CSF on DC and monocyte development. The effects of HGF were inhibited by the concurrent use of TNF. INTERPRETATION AND CONCLUSIONS: HGF appears to be a significant factor in the development of dendritic cells from CD34+ bone marrow cells.     

骨髓干细胞的可塑性及其转化为肝细胞的研究进展

干细胞(stemcell)是指一类具有自我更新能力和多向分化潜能的细胞,在特定的条件下可以分化为不同的功能细胞,形成多种组织和器官.骨髓起源的干细胞即骨髓干细胞主要有三种:造血干细胞、骨髓基质干细胞和多能前体细胞.骨髓干细胞具有可塑性(plasticity),即在一定的条件下能转化为不同于其组织来源的细胞.目前有四种机制来解释骨髓干细胞可塑性:(1)多能组织干细胞在出生后可以存在于体内多种组织和器官中,它们的增殖与分化与各自的局部环境有关;(2)成体许多组织内余存一种稀有数量的原始干细胞,其类似于胚胎干细胞,始终保持着胚胎干细胞的特性,在不同的条件刺激下能分化为内胚层、中胚层、外胚层多种组织细胞;(3)组织干细胞可以经历去分化或转分化的过程,经过基因重组,转化为不同组织来源的细胞;(4)细胞融合改变细胞特性.目前体内外试验均证实了骨髓干细胞向肝细胞的转化.骨髓干细胞在体内或体外转化为肝细胞,为临床应用开辟了新的道路.     

Placental/umbilical cord blood for unrelated-donor bone marrow reconstitution: relevance of nucleated red blood cells

Placental/umbilical cord blood (PCB) is a source of hematopoietic stem cells for bone marrow reconstitution. Engraftment speed and survival are related to the total nucleated cell (TNC) dose of the graft. This study explored the possible influence on engraftment of nucleated red blood cells (NRBCs) in the graft. Automated hematology analyzers were used to enumerate TNCs. NRBCs were counted by visual examination or by using an automated analyzer. Hematopoietic progenitor cells were enumerated as either colony-forming cells or CD34(+) cells. Transplant centers reported on transplant outcome in 1112 patients given PCB grafts through September 2001. NRBCs correlated with progenitor cell numbers. Both white blood cell and NRBC dose were independently predictive of myeloid engraftment speed. Because NRBC dose predicted engraftment speed, inclusion of NRBCs in the TNC count does not reduce the effectiveness of the prefreezing TNC count as an index of the quality of a PCB unit as a graft. The correlation between the number of NRBCs and the number of hematopoietic progenitor cells probably reflects the involvement of early stem cells in erythroid responses.     

Granulocyte/macrophage progenitor cells from peripheral blood and bone marrow differ in their response to prostaglandin E1

Prostaglandins of the E series (PGE) inhibit proliferation of normal bone marrow granulocyte/macrophage progenitors (CFU-GM). Circulating CFU-GM are known to differ from marrow CFU-GM in many characteristics, and in the present study, we compared the effect of PGE1 on circulating and bone marrow progenitors in normals and in patients with chronic myelogenous leukemia (CML). PGE1 caused a dose-dependent inhibition of normal marrow CFU-GM. Circulating CFU-GM were inhibited only at concentrations of 10(-5) mol/L or greater, and progenitor proliferation was, in fact, significantly stimulated at PGE1 concentrations between 10(-8) and 10(-6) mol/L. Bone marrow CFU-GM from patients with CML were inhibited in a manner similar to that of normal bone marrow. Circulating cells from patients with CML were, however, less sensitive to PGE1 inhibition than CML bone marrow cells and demonstrated a pattern intermediate between normal circulating and normal marrow progenitors. These studies suggest that peripheral blood and bone marrow contain different progenitor cell populations.     

Bone marrow accessory cells regulate human bone precursor cell development

OBJECTIVE: Much remains to be learned about the intimate relationship between bone marrow and its surrounding tissue: the bone. We hypothesized that bone marrow accessory cell populations might regulate the development of human bone precursor cells. MATERIALS AND METHODS: We used immunologic phenotyping, and isolation methods to fractionate subpopulations of nonadherent, low-density (NALD) human bone marrow cells. These cells were examined for their ability to support the serum-free survival, proliferation, and expression of bone proteins by highly purified populations of human bone precursor cells. Quantitative assessment of the accessory cell populations as well as human bone precursor cells phenotype was performed using multiparameter flow cytometry. Bone protein expression was evaluated by immunocytochemistry, Western analysis, and enzymatic analysis (for alkaline phosphatase activity). RESULTS: Human bone marrow contains a cell population that stimulates the development of purified bone precursor cells. Feeder-layer studies demonstrate that these osteopoietic accessory cells (OACs) do not require cell-cell interaction to promote bone precursor cell development but, rather, produce soluble molecules responsible for their effects. Flow cytometric analyses reveal that bone marrow derived B cells, T cells, macrophages, natural killer cells, and endothelial cells do not produce this stimulatory factor. The (growth) factor cannot be replaced by addition of exogenous cytokines. The isolation of human transforming growth factor beta receptor type II (TGF-betaRII)-positive cells increases OAC-specific activity in bone cell ex vivo expansion cultures. Moreover, isolation of OAC bone marrow cells characterized by high TGF-betaRII expression, relatively low cellular complexity, and small size yields a population that is highly enriched for OACs. CONCLUSION: We conclude that human bone marrow contains a population of OACs that are an obligate requirement for the early phases of bone cell development ex vivo.     

Commitment of bone marrow cells to hepatic stellate cells in mouse

BACKGROUND/AIMS: Recently, several cells found within the liver have been reported to derive from bone marrow (BM). This study sought to examine the commitment of BM cells to hepatic stellate cell (HSC) lineage in mouse liver. METHODS: We transplanted BM cells from green fluorescent protein (GFP) transgenic mice into age-matched C57BL/J mice. Hepatic nonparenchymal cells were isolated from the livers of BM-transplanted mice using density gradient centrifugation with Nycodenz. The expression of lineage markers by the isolated cells was evaluated by RT-PCR and immunostaining. We then examined the histology of liver tissues obtained from BM-transplanted mice with and without carbon tetrachloride-induced injury. RESULTS: GFP-expressing cells with intracytoplasmic lipid droplets comprised 33.4 +/- 2.3% of the cells isolated by density gradient centrifugation. These cells expressed the HSC lineage markers, such as desmin and glial fibrillary acidic protein (GFAP), by both RT-PCR and immunostaining. During a 7-day culture, GFP-positive cells began to express alpha-smooth muscle actin, a marker of activated HSC. In the liver of BM-transplanted mice, GFP-positive nonparenchymal cells expressed GFAP and extended their process around hepatocytes. Upon liver injury, these cells also co-expressed desmin and alpha-smooth muscle actin. CONCLUSIONS: Nonparenchymal cells, derived from transplanted BM, acquired HSC characteristics in both quiescent and activated states.