A population of very small embryonic-like (VSEL) CXCR4(+)SSEA-1(+)Oct-4+ stem cells identified in adult bone marrow.

By employing multiparameter sorting, we identified in murine bone marrow (BM) a homogenous population of rare (approximately 0.02% of BMMNC) Sca-1(+)lin(-)CD45- cells that express by RQ-PCR and immunohistochemistry markers of pluripotent stem cells (PSC) such as SSEA-1, Oct-4, Nanog and Rex-1. The direct electronmicroscopical analysis revealed that these cells are small (approximately 2-4 microm), posses large nuclei surrounded by a narrow rim of cytoplasm, and contain open-type chromatin (euchromatin) that is typical for embryonic stem cells. In vitro cultures these cells are able to differentiate into all three germ-layer lineages. The number of these cells is highest in BM from young (approximately 1-month-old) mice and decreases with age. It is also significantly diminished in short living DBA/2J mice as compared to long living B6 animals. These cells in vitro respond strongly to SDF-1, HGF/SF and LIF and express CXCR4, c-met and LIF-R, respectively, and since they adhere to fibroblasts they may be coisolated with BM adherent cells. We hypothesize that this population of Sca-1(+)lin(-)CD45- very small embryonic-like (VSEL) stem cells is deposited early during development in BM and could be a source of pluripotent stem cells for tissue/organ regeneration.     

Differentiation pathways of pluripotent hemopoietic stem cells in long-term mouse bone marrow culture

Differentiation pathways of CFU-S were examined in mouse bone marrow long-term culture system. E/G ratios of non-adherent CFU-S in this system increased from 2 to 7-9 by 2-3 weeks of culture and then fluctuated during the culture period. On the other hand, E/G ratios of adherent CFU-S were lower than that of non-adherent CFU-S, and always remained under 2. Some of the supernatants collected from culture flasks at weekly intervals increased E/G ratios of normal bone marrow CFU-S after 18-20 h of incubation. These results suggest that the differentiation of CFU-S is controlled by humoral factors, secreted from yet unknown cells in bone marrow, in this system.     

The tumorigenicity of human embryonic and induced pluripotent stem cells

The unique abilities of human pluripotent stem cells to self-renew and to differentiate into cells of the three germ layers make them an invaluable tool for the future of regenerative medicine. However, the same properties also make them tumorigenic, and therefore hinder their clinical application. Hence, the tumorigenicity of human embryonic stem cells (HESCs) has been extensively studied. Until recently, it was assumed that human induced pluripotent stem cells (HiPSCs) would behave like their embryonic counterparts in respect to their tumorigenicity. However, a rapidly accumulating body of evidence suggests that there are important genetic and epigenetic differences between these two cell types, which seem to influence their tumorigenicity.     

A novel tumor cell line cloned from mutated human embryonic bone marrow mesenchymal stem cells

A novel tumor cell line, denominated F6, was established from mutated human embryonic bone marrow mesenchymal stem cells (MSCs) which were induced by the GM-CSF and IL-4 in vitro. The characteristics of the F6 cell line, such as surface antigens, cell cycle, growth curve, gene expression, morphology, cytogenetics and tumor model were analyzed. The F6 cells were round and grew suspended in a plastic dish. The cell line has a strong self-renewal capability, was positive for CD13, CD29, CD44, but negative for CD1alpha, CD3, CD10, CD14, CD23, CD33, CD34, CD38, CD41, CD45, CD54 and HLA-DR. The surface antigens were lower than those of human embryonic MSCs. The karyotype of F6 cells was abnormal. The cell cycle included: G0/G1 phase, 52.24%; G2/M phase, 8.00%; S phase, 41.76%. After the cells had been passaged serially for more than 17 months (62 passages), their characteristics were still retained. The F6 cells resulted in tumors in SCID nude mice in vivo (8/8) and caused metastasis (3/8). The pathologic examination revealed that the tumor cells extensively invaded surrounding normal tissues such as dermis, muscular tissue, nerve tissue, adipose tissue and lymphoid tissue. F6 cell line, tumor tissues derived from F6 cells and the MSCs expressed different levels of the nucleostemin gene. These findings suggested that F6 may be a novel tumor cell line. It may provide evidence for the theory that cancer originates from stem cells, and may be useful for the investigation on safety of human MSCs in the clinical application.     

The comparison of biologic characteristics between mice embryonic stem cells and bone marrow derived dendritic cells

OBJECTIVE This research was to induce dendritic cells （DCs） from mice embryonic stem cells and bone marrow mononuclear cells in vitro, and then compare the biologic characteristics of them. METHODS Embryonic stem cells （ESCs） suspending cultured in petri dishes were induced to generate embryonic bodies （EBs）. Fourteen-day well-developed EBs were transferred to histological culture with the same medium and supplemented 25 ng/ml GM- CSF and 25 ng/ml IL-3. In the next 2 weeks, there were numerous immature DCs outgrown. Meantime, mononuclear cells isolated from mice bone marrow were induced to derive dendritic cells by supplementing 25 ng/ml GM-CSF and 25 ng/ml IL-4, and then the morphology, phenotype and function of both dendritic cells from different origins were examined. RESULTS Growing mature through exposure to lipopolysaccharide （LPS）, both ESC-DCs and BM-DCs exhibited dramatic veils of cytoplasm and extensive dendrites on their surfaces, highly expressed CD11c, MHC-II and CD86 with strong capacity to stimulate primary T cell responses in mixed leukocyte reaction （MLR）. CONCLUSION ESC-DC has the same biologic characteristics as BM-DC, and it provides a new, reliable source for the functional research of DC and next produce corresponding anti-tumor vaccine.     

Expression of embryonic stem cell marker Oct-4 and its prognostic significance in rectal adenocarcinoma

Objective: Recent evidence suggests that Oct-4 is highly expressed in several cancers, and its expression contributes to tumor growth. In this study, we investigated the level of Oct-4 expression in rectal adenocarcinoma, and evaluated the prognostic significance of Oct-4 expression in these cases.Methods: The immunohistochemical expression of Oct-4 was evaluated in 52 formalin-fixed paraffin-embedded postoperative rectal adenocarcinoma tissue samples. The impact of the immunoreactivity of Oct-4 in regard to clinical outcome was determined by Kaplan-Meier and log-rank.Results: The expression level of Oct-4 ranged from 0 to 18.5%. There was no significant association between Oct-4 expression and gender (P=0.772), age (P=0.123), clinical stage (P=0.391), and histological grade (P=0.056). The 3-year local recurrence-free rates with negative and positive expression of Oct-4 were 83.5% and 75.0%, respectively (P=0.583). The 3-year metastasis-free rates with negative and positive expression of Oct-4 were 88.6% and 61.9%, respectively (P=0.035). The 3-year overall survival rates with negative and positive expression of Oct-4 were 77.9% and 49.0%, respectively (P=0.037).Conclusion: The results suggest that embryonic stem cell marker Oct-4 expression may have prognostic significance in patients with rectal adenocarcinoma. However, to confirm this more and larger studies are required.     

Isolation and characterization of normal adult human epithelial pluripotent stem cells

Both reproductive and therapeutic cloning of human stem cells have been made possible with recent technological advances in the isolation of embryonic stem cells and of pluripotent stem cells from adult tissues. We have isolated normal human kidney and human breast epithelial stem cells, as well as having characterized "immortalized" cells from human neuronal and human pancreatic tissue (Trosko et al., Methods 20:245-264, 2000). The isolation was motivated by the stem cell theory of carcinogenesis. Based on the assumption that stem cells would not express connexin genes, nor have functional gap junctional intercellular communication (GJIC), we have demonstrated that the human kidney, breast, neuronal, and pancreatic stem cells can divide either symmetrically or asymmetrically, depending on whether they are grown in microenvironmental conditions that suppress GJIC (the undifferentiated, proliferative state) or induce GJIC (the differentiated state). Normal breast epithelial stem cells appear to be intrinsically "immortal" until induced to express GJIC, at which time, with appropriate substrate and microenvironmental nutrients, they can form three-dimensional "organoids." expressing markers associated with the mature mammary tissue and forming a physical structure very similar to the in vivo budding, ductal structures. The breast stem cells can be prevented from "mortalizing" and can be converted to neoplastic cells, which maintain many phenotypes of the stem cells.     

Recovery of blood and bone marrow stem cells following intense chemotherapy and autologous bone marrow transplantation

Sixteen patients with advanced (stage III) malignant melanoma were treated with escalating doses of intravenous BCNU and melphalan starting at 400 and 35 mg/m2, respectively, and escalating to 1,000 and 110 mg/m2, respectively, combined with autologous marrow transplantation. The duration of granulocytopenia and time to granulocyte recovery was similar in all groups regardless of chemotherapy dose. Platelet recovery was delayed in patients receiving the highest doses of chemotherapy. This study showed that bone marrow colony-forming units in culture took as long as 6 months to recover. This was adequate to bring peripheral blood counts to normal but not to pretreatment levels. These studies indicate that autologous bone marrow transplantation is beneficial in enhancing short-term recovery, but may not be beneficial in the long-term hematopoietic recovery.     

Early disappearance of murine plasmocytoma stem cells in long-term bone marrow culture.

Long-term bone marrow culture (LTBMC) was evaluated as a purging procedure in the murine plasmocytoma MOPC-315s system. MOPC-315s cells injected in Balb-c mice rapidly proliferate both in marrow and spleen, where macroscopic tumor colonies develop. A linear relationship between the number of injected cells and spleen colonies was observed, consistent with the presence of 1 clonogenic myeloma stem cell out of 1800 cells. In vitro, MOPC-315s cells are easily identifiable as rosette-forming cells (RFC+) with trinitrophenil acid (TNP) coated sheep red blood cells. When bone marrow (BM) cells containing 20-40% RFC+ were seeded in LTBMC, RFC+ rapidly decreased and were no longer detectable by day 14 of culture. Clonal Ig gene rearrangement was evident at time 0, but it was no more detectable later on. In addition, cells taken at days 14 and 21 of culture were no more tumorigenic when injected in vivo. The results suggest the efficacy of the LTBMC for the in vitro elimination of myeloma cells, including the neoplastic stem cells.     

神经胶质细胞神经干细胞和肿瘤干细胞之间依赖微生态环境而转化的假设

我们于20世纪70年代开始进行人脑胶质瘤的研究,建立了至今仍在应用的中国第一株人脑胶质瘤体外细胞系SHG44及其裸小鼠皮下移植瘤模型NHG-1~([1-2]),並在此平台上完成了抗人脑胶质瘤单克隆抗体制备~([3])、生物导向诊治~([4])、人脑胶质瘤细胞诱导分化~([5-6])和人脑胶质瘤干细胞研究等11个国家自然科学基金资助项目(39070816、39370684、39670735、39870862、30070772、30371457、30371457,30400457、30672164、30772241和30872654).     

A case of retroperitoneal neuroblastoma in an adult with extensive bone marrow metastasis

We present a case of retroperitoneal neuroblastoma in a 27-year-old male with extensive bone marrow metastasis at the first presentation. After the simple excision of the tumor, adjuvant multi-drug chemotherapy, consisting of vincristine, actinomycin-D, ifosfamide, doxorubicin, carboplatin and etoposide, was carried out for 17 months, leading to complete remission. Ten months after completion of the chemotherapy, the tumor recurred with bone marrow metastasis. He further developed thoracic vertebral metastases resulting in paraplegia, and died of the disease 41 months after the presentation. The clinical course of this case, with its emphases especially on the effect of the chemotherapy, is described in this report. Since the clinical characteristics and treatment strategies for adult neuroblastoma have not yet been well established, they remain to be investigated in detail.     

Oct-4在肿瘤组织中的表达及其临床意义的研究现状

参考文献.结果:转录因子Oct-4属POU家族蛋白,在动物早期胚胎发育过程中起着重要作用,其通过激活或抑制下游靶基因来参与维持细胞的多向潜能性及未分化状态.已在多种实体肿瘤细胞中发现Oct-4异常高表达,由此更进一步证实了肿瘤干细胞学说.Oct-4的表达可能与肿瘤的恶性程度及生物学行为、肿瘤放化疗抵抗密切相关.结论:有关Oct-4的研究可能为判断肿瘤预后提供新的指标,也有利于发展新的抗癌药物作用靶点.     

Behaviour of heat-treated bone marrow cells in long-term bone marrow cultures.

The effect of prior heat treatment on the ability of bone marrow cells to form long-term bone marrow culture has been studied. Bone marrow cells were heated for various times in the temperature range of 39-43 degrees C and then cultured in the modified Dexter type suspension culture. At weekly intervals, the behaviour of the cultures in terms of stroma formation and confluency, cellular viability, and myelopoiesis were evaluated. The results show that there was a dose-dependent decrease in the number of viable cells in the non-adherent fraction of the cultures. Cytological analysis of these cells showed a strong shift towards macrophage population in the successive weeks of the cultures and also as a function of heat dose delivered to these cultures. The stroma formation was delayed or inhibited as a function of the heat dose. The number of granulocyte-macrophage colony forming cells (CFU-GM) in both adherent and non-adherent fraction of the cultures were decreased substantially after hyperthermia treatment. At 41 degrees C and higher temperatures, the CFU-GM were severely diminished in both fractions. The dose response experiments showed that the decrease in the number of CFU-GM was dependent on the heat dose. The results suggest that CFU-GM is an extremely sensitive target in the hyperthermia treatment of bone marrow cells and heat-treated bone marrow cells lose their ability to maintain long-term cultures.     

骨髓间充质干细胞多向分化潜能和微环境关系的研究

目的：深入研究骨髓间充质干细胞（mesenchymal stem cells,MSCs）多向分化的潜能与微环境的关系,从而为促进MSCs向目标组织细胞诱导分化创建新的实验方法。方法：大鼠MSCs进行分离,体外培养、扩增、鉴定。将MSCs向脂肪细胞和胰岛细胞方向诱导分化,并深入研究在不同的微环境下,其分化能力的差别,对照组诱导剂为含有角朊细胞生长因子（KGF）、胰岛素-转铁蛋白-硒（ITS）、尼克酰胺的无血清DMEM／F12培养基,实验组在对照组基础上添加胰腺条件培养液;对诱导的胰岛细胞进行观察、双硫腙染色,并进行葡萄糖刺激实验,测定细胞分泌胰岛素及C-肽功能。结果：培养的MSCs表现为非造血干细胞特性。其可向脂肪细胞,胰岛细胞等不同组织细胞分化。对照组和实验组均可分化为胰岛细胞,但实验组分化而成的胰岛细胞在数量和功能上均高于对照组。结论：MSCs具有多向分化潜能,其分化能力在特定的微环境下更强。     

骨髓间充质干细胞多向分化潜能和微环境关系的研究

目的:深入研究骨髓间充质干细胞(mesenchymal stem cells,MSCs)多向分化的潜能与微环境的关系,从而为促进MSCs向目标组织细胞诱导分化创建新的实验方法。方法:大鼠MSCs进行分离,体外培养、扩增、鉴定。将MSCs向脂肪细胞和胰岛细胞方向诱导分化,并深入研究在不同的微环境下,其分化能力的差别,对照组诱导剂为含有角朊细胞生长因子(KGF)、胰岛素-转铁蛋白-硒(ITS)、尼克酰胺的无血清DMEM/F12培养基,实验组在对照组基础上添加胰腺条件培养液;对诱导的胰岛细胞进行观察、双硫腙染色,并进行葡萄糖刺激实验,测定细胞分泌胰岛素及C-肽功能。结果:培养的MSCs表现为非造血干细胞特性。其可向脂肪细胞,胰岛细胞等不同组织细胞分化。对照组和实验组均可分化为胰岛细胞,但实验组分化而成的胰岛细胞在数量和功能上均高于对照组。结论:MSCs具有多向分化潜能,其分化能力在特定的微环境下更强。     

The separation of a mixture of bone marrow stem cells from tumor cells: An essential step for autologous bone marrow transplantation

KHT tumor cells were mixed with mouse bone marrow to simulate a sample of bone marrow containing metastatic tumor cells. This mixture was separated into a bone marrow fraction and a tumor cell fraction by centrifugal elutriation. Elutriation did not change the transplantability of the bone marrow stem cells as measured by a spleen colony assay and an in vitro erythroid burst forming unit assay. The tumorogenicity of the KHT cells was similarly unaffected by elutriation. The data showed that bone marrow cells could be purified to less than 1 tumor cell in more than 10⁶ bone marrow cells. Therefore, purification of bone marrow removed prior to lethal radiation-drug combined therapy for subsequent autologous transplantation appears to be feasible using modifications of this method if similar physical differences between human metastatic tumor cells and human bone marrow cells exist. This possibility is presently being explored.     

Bone marrow mesenchymal stem cells provide an alternate pathway of osteoclast activation and bone destruction by cancer cells

The bone is the third most common site of cancer metastasis. To invade the bone, tumor cells produce osteoclast-activating factors that increase bone resorption by osteoclasts. Here we report that human neuroblastoma cells that form osteolytic lesions in vivo do not produce osteoclast-activating factors but rather stimulate osteoclast activity in the presence of human bone marrow mesenchymal stem cells. This alternative pathway of osteoclast activation involves a nonadhesive interaction between neuroblastoma cells and bone marrow mesenchymal stem cells. Stimulated bone marrow mesenchymal stem cells express markedly increased levels of interleukin-6, which is then responsible for osteoclast activation. This report describes a critical role of bone marrow mesenchymal stem cells in bone destruction in cancer.     

RNA-based gene transfer for adult stem cells and T cells.

Electroporation of mRNA has become an established method for gene transfer into dendritic cells for immunotherapeutic purposes. However, many more cell types and applications might benefit from an efficient mRNA-based gene transfer method. In this study, we investigated the potential of mRNA-based gene transfer to induce short-term transgene expression in adult stem cells and activated T cells, based on electroporation with mRNA encoding the enhanced green fluorescent protein. The results show efficient transgene expression in CD34-positive hematopoietic progenitor cells (35%), in in vitro cultured mesenchymal cells (90%) and in PHA-stimulated T cells (50%). Next to presentation of gene transfer results, potential applications of mRNA-based gene transfer in stem cells and T cells are discussed.