Growth of Human Bone Marrow in Liquid Culture

A system for the cultivation of normaland malignant human bone marrowcells in liquid medium is described.The apparatus used is an in vitro diffusion chamber in which cells growin suspension and upon a dialysismembrane. Proliferation and maturation of normal granulocytes and macrophages were sustained in culture forseveral weeks in the absence of exogenous stimulating substances. Cellreplication was documented by a risein viable counts, ³H-thymidine incorporation, and labeled mitoses. Theentire maturational sequence of thegranulocyte and macrophage serieswas identified in culture, and the cellswere characterized by light and electron microscopy, cytochemical properties, phagocytic ability, and the presence of surface receptors for IgG.Nucleated red cell precursors wereobserved in some cultures for up to 11days. Malignant cells from patientswith various hematologic neoplasmswere also successfully cultured. Withthis system human myeloma cells weremaintained in culture for up to 3 wkon primary explant, and continued tosynthesize immunoglobulin. The invitro diffusion chamber technique permits the cultivation of normal humanbone marrow cells in liquid mediumand provides a convenient means forstudying normal and neoplastichematopoietic cell differentiation andfunction in short-term culture.

Submitted on May 29, 1972 Revised on June 13, 1972 Accepted on June 20, 1972     

Hypothesis: Engrafted Human Bone Marrow and Blood Cells in Culture

Lymphoblasts appearing in immunosuppressed patients after bone marrow transfusion are compared to thosethat can be established in vitro as permanent lymphoid cell lines. It is suggested that Epstein-Barr virus (EBV)could be responsible for the recurrent "lymphoblastic leukemia" inthese patients and that the transplanted cells may be a clone of nonmalignant cells that has becomecapable of growing without normalrestraints. It is important that in futurepatients the transplanted cells be characterized as to morphology, chromosome constitution, relative clonabilityand transplantability, the presence ofEBV, T or B cell-like traits, and theirgrowth potential in immunosuppressedheterologous hosts. The antibody titerto EBV should be measured beforeand after leukocyte transfusion.

     

Platelet-Derived Growth Factor Enhances Granulopoiesis Via Bone Marrow Stromal Cells

Platelet-derived growth factor (PDGF), a growth factor for connective tissue cells, stimulates erythropoiesis and megakaryocytopoiesis in vitro but the effect of PDGF on granulocyte proliferation remains unknown. The effect of the recombinant human PDGF-BB isoform on granulopoiesis was investigated in this study. The results show that PDGF significantly stimulated murine colony-forming unit-granulocyte-monocyte (CFU-GM) proliferation in a dose-dependent manner (1 to 100 ng/mL) using murine bone marrow cells (n = 4). Maximum stimulation was obtained with 50 ng/mL of PDGF (P < .01). The effect of PDGF on murine CFU-GM proliferation was compared with that of interleukin (IL)-3, IL-6, granulocyte-monocyte colony-stimulating factor (GM-CSF), and acidic fibroblast growth factor (aFGF) at their optimal doses. The stimulating activity of PDGF was higher than that of aFGF but lower than that of IL-3, IL-6, or GM-CSF. There is no synergistic effect between PDGF and IL-3 or IL-6, but a significant enhancing effect was observed in IL-3 plus IL-6. PDGF also stimulated the growth of CFU-GM with CFU-megakaryocyte in the presence of bone marrow stromal cells. We also found that PDGF had similar a effect on human CFU-GM proliferation using bone marrow mononuclear cells (MNC). However, the increase in PDGF-stimulated CFU-GM proliferation was inhibited by anti-GM-CSF, anti-IL-3, and anti-IL-6 antibodies (n = 4), suggesting that endogenously produced GM-CSF, IL-3, and IL-6 may play a role in the PDGF-induced CFU-GM proliferation. Furthermore, PDGF (1 to 100 ng/mL) did not show any effect on CFU-GM proliferation when replacing bone marrow MNC with immunomagnetic selection-enriched CD34+ cells from human cord blood (n = 5; purity, 91% +/- 6.5%). This study indicates that PDGF may indirectly enhance CFU-GM proliferation by inducing the bone marrow stromal cells to produce GM-CSF, IL-3, or IL-6.     

大鼠血管内皮祖细胞培养及鉴定

目的探索骨髓来源的血管内皮祖细胞的分离培养方法,为缺血性疾病治疗找到新的移植细胞来源。方法采集SD雄性大鼠骨髓细胞,用梯度密度离心法分离单核个细胞,种植于提前包埋了纤维连接蛋白的培养皿中培养,将血管内皮生长因子(VEGF)、重组纤维细胞生长因子(bFGF)和重组上皮生长因子(EGF)作为诱导剂,将72h后贴壁细胞(A组)和非贴壁细胞(B组)分别培养,观察细胞形态的变化以及数量变化,取A组在第14天,应用免疫组化和免疫荧光技术鉴定内皮祖细胞系列标志。结果A组细胞经过体外诱导后大量扩增,在28d时仍有强大扩增的活力,呈铺路石样,而B组细胞短期内可以大量增殖,呈长索样,之后逐渐变少;在细胞迁移实验中,A组细胞可以相互融合形成血管结构,而B组细胞之间则不能融合,随着时间推移,逐渐凋亡;A组经过CD34免疫荧光、CD133、FLK-1免疫组化鉴定呈阳性,并能特异性吸附FITC-UEA-和内吞DIL-Ac-LDL。结论自体骨髓细胞通过贴壁换液后可以分离培养出内皮祖细胞,取贴壁细胞经过体外诱导后大量扩增,并通过表面标记物鉴定可以确认为内皮祖细胞,而非贴壁细胞不能大量增殖,故将贴壁分离方法来筛选内皮祖细胞,此方法简易,并能满足细胞移植的需要,因而,在移植治疗脑缺血引起的内皮损伤应该有较好的临床应用前景。     

The Growth of Human Bone Marrow in Diffusion Chambers

S ummary . Diffusion chambers containing normal human bone marrow were implanted intraperitoneally into normal and irradiated mice and cultured for various periods. Granulocytes, macrophages and lymphocytes comprised the majority of the cells harvested in all cultures. The most impressive cell growth occurred when the chambers were implanted into primary hosts irradiated with 840 R and then retransplanted on Day 8 of the culture into secondary hosts irradiated with the same dose. Linear relationships were found between the number of nucleated cells inoculated into the chambers and the number of cells harvested after 8 or 10 days of culture in heavily irradiated hosts.     

Schwann Cells of the Bone Marrow

Nerves containing numerous Schwanncells can be found in bone marrow withroutine histological methods. Thin nervebundles and single nerve fibers containing Schwann cells can be identified histologically with help of methods whichdemonstrate nerve fibers and myelin.Smears of marrow stained with the May-Grünwald-Giemsa method are not adequate for demonstration of nerve fibers,but their satellite Schwann cells appearwell stained. Histological, cytologicaland ultrastructural characteristics of thesecells are described in detail. Blood-forming cells and nerve fibers with theirSchwann cells lie in close proximitythroughout the marrow. This circumstance suggests that interaction may takeplace between both elements.

Submitted on January 23, 1970 Accepted on February 24, 1970     

Apparent C Trisomy in Bone Marrow Cells

A supernumerary chromosome belonging to group C was encountered in bone marrow cells from two patients. One patient, suffering from an atypical myeloproliferative syndrome resembling chronic myeloid leukaemia, has had the apparent C trisomy in 100% of his dividing bone marrow cells for over a year. The other patient, who died of acute myelomonocytic leukaemia, had a mixture of normal and apparently C trisomic cells in his bone marrow. In both instances, phytohaemagglutininstimulated lymphocytes as well as fibroblasts of skin origin showed normal karyotypes. Clinical investigations, including cytochemical characterization of abnormal cells and quantitation and fractionation of vitamin B₁₂-binding proteins, are reported. The results failed to reveal any common clinical entity. Previous case reports are reviewed. The trisomic condition has been discovered in disorders affecting the myeloid, monocytoid and erythroid, but not the lymphoid, cells. The significance of C trisomy in haematological disorders remains obscure.     

Bone Marrow Cells and Myocardial Regeneration

Hematopoietic stem cell (HSC) plasticity and its clinical application have been studied profoundly in the past few years. Recent investigations indicate that HSC and other bone marrow stem cells can develop into other tissues. Because of the high morbidity and mortality of myocardial infarction and other heart disorders, myocardial regeneration is a good example of the clinical application of HSC plasticity in regenerative medicine. Preclinical studies in animals suggest that the use of this kind of treatment can reconstruct heart blood vessels, muscle, and function. Some clinical study results have been reported in the past 2 years. In 2003, reports of myocardial regeneration treatment increased significantly. Other studies include observations on the cell surface markers of transplanted cells and treatment efficacy. Some investigations, such as HSC testing, have focused on clinical applications using HSC plasticity and bone marrow transplantation to treat different types of disorders. In this review, we focus on the clinical application of bone marrow cells for myocardial regeneration.     

Peripheral Blood Cells from Patients with Aplastic Anaemia in Partial Remission Suppress Growth of their Own Bone Marrow Precursors in Culture

S ummary In 12 patients with severe aplastic anaemia who had achieved self-sustaining autologous bone marrow function after treatment with antilymphocyte globulin, or with cyclophosphamide given for attempted bone marrow transplantation, colony formation by all haemopoietic precursors remained far below normal. Precursors from peripheral blood, erythroid precursors in particular, failed to form a normal number of colonies. This paucity of colony formation does not reflect a true lack of precursor cells but is due to circulating cells which impair maturation. Addition of low density peripheral blood cells to autologous bone marrow cultures diminished colony formation by granulocyte-macrophage precursors (GM-CFC) and abolished 'burst' formation by BFU-E. Strong auto-inhibition preceded relapse in four of eight patients. The phenomenon was not observed in five normals, in five aplastic anaemia patients with stable haemopoietic grafts and in three polytransfused control patients.
The T-cell poor subpopulation of peripheral blood cells, containing mainly B-cells and macrophages, was especially inhibitory. Accordingly, removal of plastic adherent cells from bone marrow cell suspensions improved plating efficiency in aplastic anaemia patients, but not in normals. Isolated E-rosette positive cells had no negative effect.
Inhibition could only be demonstrated in the autologous situation. Colony formation by normal allogeneic peripheral blood precursors was not impaired by patient cells. The phenomenon is likely to reflect residual disease activity which is compensated in vivo but can be demonstrated in vitro. It may be of help in early recognition of patients who are at risk of relapse after autologous bone marrow reconstitution.     

不同诱导条件对人骨髓基质干细胞向内皮分化的影响

目的探讨不同诱导条件对人骨髓基质干细胞向内皮分化的影响。方法采用密度梯度离心法分离培养人骨髓基质干细胞,用荧光激活细胞分选法分析骨髓基质干细胞CD34、CD105和CD166的表达率;用免疫荧光细胞化学法观察骨髓基质干细胞在细胞因子(50μgL血管内皮细胞生长因子、5μgL碱性成纤维细胞生长因子、100mgL内皮细胞生长补充因子)、内皮条件培养基或与成熟兔主动脉内皮共培养5天时vWF或和Flk1蛋白的表达。结果分离培养的骨髓基质干细胞CD34表达率为4.16%±0.16%,与阴性对照(4.06%±0.23%)相比无统计学差异,CD105表达率为90.20%±2.35%,CD166表达率为82.30%±3.22%,均明显高于阴性对照(P<0.05)。诱导前骨髓基质干细胞不表达vWF和Flk1蛋白,经血管内皮细胞生长因子、碱性成纤维细胞生长因子和内皮细胞生长补充因子等细胞因子联合诱导5天时,33.42%骨髓基质干细胞开始表达vWF蛋白;与成熟内皮共培养5天时,vWF染色仍为阴性,但25.71%骨髓基质干细胞开始表达Flk1;用内皮条件培养基培养5天时,骨髓基质干细胞vWF和Flk1染色均为阴性。结论细胞因子和成熟内皮细胞均能诱导骨髓基质干细胞向内皮分化,而内皮条件培养基不能诱导骨髓基质干细胞向内皮分化。     

采用1.068 g/ml分离液对犬骨髓间充质干细胞的分离纯化与体外培养方法的探讨

目的探讨更为可靠、纯净度高的犬骨髓间充质于细胞分离、培养方法。方法将犬骨髓抽取液分别以1．068g／ml及1．073g／ml分离液进行密度梯度离心，采用贴壁培养法获得骨髓间充质干细胞（MSCs）；以LG-DMEM加15％FBS培养；应用于细胞表面标志蛋白，多向诱导分化等方法进行细胞鉴定。结果采用1．068g／ml分离液可获得纯度较高的单核细胞，接种细胞生长良好，孵育24h即可见细胞贴壁生长，平均倍增周期为1d，细胞呈纺锤状，螺旋梳状排列。连续培育8代以上，未见细胞形态、增殖特性改变。MSCs表面标志SH2阳性，CD45阴性，可定向分化为心肌细胞、成骨细胞及脂肪细胞。结论采用1．068g／m1分离液能够较好地进行犬MSCs的体外分离、培养与扩增，分离得到的细胞具备MSCs的特性。     

Thymidine Kinase Activity in Human Bone Marrow Cells

The thymidine kinase activity per 10⁶ DNA-synthesising marrow cells and the rate of incorporation of tritiated thymidine into the DNA of 10³ DNA-synthesising marrow cells were estimated in 9 haematologically normal patients and 49 patients suffering from a variety of haematological disorders. Slight increases in thymidine kinase activity were found in 6 of the 31 patients with haematological diseases associated with normoblastic erythropoiesis and greater increases were found in 3 of the 18 patients with megaloblastic haemopoiesis due to vitamin B₁₂ or folate deficiency. In the latter group, there was a statistically significant inverse correlation between haemoglobin levels and thymidine kinase activity. No correlation was found between thymidine kinase activity and the rate of incorporation of tritiated thymidine in either the normoblastic or megaloblastic group, suggesting that the level of thymidine kinase activity does not limit the rate of incorporation of exogenously supplied thymidine into the DNA of human bone marrow cells.     

骨髓基质干细胞与心脏疾病

各种心脏疾病的临床治疗仍是困扰着临床医师的一大难题,目前针对心脏疾病的药物、介入和手术治疗的效果并不是很理想,而近年来,骨髓基质干细胞在心脏疾病方面应用成为一个新的研究亮点。现就骨髓基质干细胞移植在心脏疾病方面应用作一综述。     

骨髓间充质干细胞体外诱导分化为心肌细胞

目的观察5-氮杂胞苷(5-aza)体外诱导分化骨髓间充质干细胞(Mesenchymal Stem Cells,MSCs)成为心肌细胞的作用,并为进一步体内研究而进行细胞标记.方法提取小型猪骨髓20ml,体外分离培养骨髓间充质干细胞,分为对照组和诱导组,其中诱导组在培养第3天加入5-aza(10 μmol/L),分别在培养2周和4周时观察两组细胞光镜、电镜下形态,4周时进行磷钨酸-苏木素染色(PTAH)和肌动蛋白(actin)免疫组化染色.诱导组MSCs加入5-溴脱氧尿嘧啶(BrdU,5μg/ml),分别在作用后第2周和第4周,利用BrdU单克隆抗体免疫组化检测BrdU标记方法.此外,用含有LacZ基因的复制缺陷腺病毒转染诱导组MSCs,利用X-gal染色检测LacZ基因表达的半乳糖苷酶,观察诱导分化后MSCs的体外标记情况.结果体外能够成功地分离骨髓MSCs,5-aza诱导分化2周和4周后的MSCs在光镜下明显成为梭形,形态类似肌细胞.电镜下诱导分化后的MSCs在2周时出现肌丝结构,4周时肌丝明显增多.PTAH染色显示超过70%的细胞具有横纹肌的染色性质,actin免疫组化染色显示超过80%的诱导后MSCs肌动蛋白呈阳性表达.体外BrdU单克隆抗体检测BrdU掺入到MSCs细胞核中,X-gal染色证明LacZ基因能够进入MSCs并表达,两种体外标记方法在标记2周和4周时都为阳性.结论5-aza能够诱导骨髓MSCs成为心肌细胞,BrdU掺入和含有LacZ基因的复制缺陷腺病毒转染能够成功对诱导分化后MSCs进行标记,并且标记持续至少4周.     

Separation of Normal Mature Bone Marrow Plasma Cells

S ummary A three-phase purification process for the separation of normal bone marrow plasma cells is described. Material was obtained from haematologically normal humans and baboons. Known monoclonal and polyclonal B cell activators were avoided both in vivo and in vitro. In Phase I, bone marrow fragments were separated from cell suspensions by buoyant density methods. Marrow fragments were shown to be richer in plasma cells than the corresponding marrow cell suspensions. Phase II consisted of culturing fragments by a simple suspension method in which a selective affinity of plasma cells and marrow stromal cells resulted in further concentration of plasma cells with discharge of haemopoietic elements. Maximum concentration of plasma cells occurred within 7 d. In phase III, fragments were disaggregated with trypsin, and the marrow stromal cells were removed by their adherence properties. The resulting non-adherent fraction comprised approximately 85% plasma cells. The process allows for direct quantitative evaluation of normal plasma cell physiology in vitro.     

Separation of Human Bone Marrow Cells by Sedimentation

Sedimentation at unit gravity of human bone marrow cells, for 15 h at 4° C on a linear density gradient of Ficoll in culture medium ranging from 1.020 to 1.065 g/ml shows that a differential migration of the bone marrow cell sub-populations exists with precise mean densities 1.021 ±lx 10^?3 g/ml for lymphocytes; 1.024 ± 2.5 times 10^?3 g/ml for non-eosinophil granulocytes; 1.025 ± 2.5 times 10^?3 g/ml for metamyelocytes; 1.030 ± 3.5 times 10^?3 g/ml for other myeloid cells (myeloblasts, promyelocytes, myelocytes); 1.040 ± 3 times 10^?3 g/ml for eosinophil granulocytes; and 1.055 ± 10 times 10^?3 g/ml for megakaryocytes. The highest percentages of S phase cell and G2 and M phase cells determined by a cytofluorograph correspond to the peaks of immature myeloid cells (myeloblasts, promyelocytes and myelocytes). This method of bone marrow cell separation may be used to study the cell cycle in pathological bone marrows (leukaemia in particular) and to determine the effects and the efficiency of some antimitotics.