Isolation and characterization of neural stem cells from human fetal striatum

This paper described that neural stem cells (hsNSCs) were isolated and expanded rapidly from human fetal striatum in adherent culture. The population was serum- and growth factor-dependent and expressed neural stem cell markers. They were capable of multi-differentiation into neurons, astrocytes, and oligodendrocytes. When plated in the dopaminergic neuron inducing medium, human striatum neural stem cells could differentiate into tyrosine hydroxylase positive neurons. hsNSCs were morphologically homogeneous and possessed high proliferation ability. The population doubled every 44.28h and until now it has divided for more than 82 generations in vitro. Normal human diploid karyotype was unchanged throughout the in vitro culture period. Together, this study has exploited a method for continuous and rapid expansion of human neural stem cells as pure population, which maintained the capacity to generate almost fifty percent neurons. The availability of such cells may hold great interest for basic and applied neuroscience.     

Cancer stem cell-like cells derived from malignant peripheral nerve sheath tumors

This study aims to examine whether or not cancer stem cells exist in malignant peripheral nerve sheath tumors (MPNST). Cells of established lines, primary cultures and freshly dissected tumors were cultured in serum free conditions supplemented with epidermal and fibroblast growth factors. From one established human MPNST cell line, S462, cells meeting the criteria for cancer stem cells were isolated. Clonal spheres were obtained, which could be passaged multiple times. Enrichment of stem cell-like cells in these spheres was also supported by increased expression of stem cell markers such as CD133, Oct4, Nestin and NGFR, and decreased expression of mature cell markers such as CD90 and NCAM. Furthermore, cells of these clonal S462 spheres differentiated into Schwann cells, smooth muscle/fibroblast and neurons-like cells under specific differentiation-inducing cultural conditions. Finally, subcutaneous injection of the spheres into immunodeficient nude mice led to tumor formation at a higher rate compared to the parental adherent cells (66% versus 10% at 2.5 × 10(5)). These results provide evidence for the existence of cancer stem cell-like cells in malignant peripheral nerve sheath tumors.     

Differentiation profile of brain tumor stem cells： a comparative study with neural stem cells

Understanding of the differentiation profile of brain tumor stem cells （BTSCs）, the key ones among tumor cell population, through comparison with neural stem cells （NSCs） would lend insight into the origin of glioma and ultimately yield new approaches to fight this intractable disease. Here, we cultured and purified BTSCs from surgical glioma specimens and NSCs from human fetal brain tissue, and further analyzed their cellular biological behaviors, especially their differentiation property. As expected, NSCs differentiated into mature neural phenotypes. In the same differentiation condition, however, BTSCs exhibited distinguished differences. Morphologically, cells grew flattened and attached for the first week, but gradually aggregated and reformed floating tumor sphere thereafter. During the corresponding period, the expression rate of undifferentiated cell marker CD 133 and nestin in BTSCs kept decreasing, but 1 week later, they regained ascending tendency. Interestingly, the differentiated cell markers GFAP and β-tubulinlII showed an expression change inverse to that of undifferentiated cell markers. Taken together, BTSCs were revealed to possess a capacity to resist differentiation, which actually represents the malignant behaviors of glioma.     

Acridine yellow G blocks glioblastoma growth via dual inhibition of epidermal growth factor receptor and protein kinase C kinases

Amplification of the epidermal growth factor receptor (EGFR), frequently expressed as a constitutively active deletion mutant (EGFRvIII), occurs commonly in glioblastoma multiformes (GBM). However, blockade of EGFR is therapeutically disappointing for gliomas with PTEN deletion. To search for small molecules treating this aggressive cancer, we have established a cell-based screening and successfully identified acridine yellow G that preferentially blocks cell proliferation of the most malignant U87MG/EGFRvIII cells over the less malignant U87MG/PTEN cells. Oral administration of this compound markedly diminishes the brain tumor volumes in both subcutaneous and intracranial models. It directly inhibits EGFR and PKCs with IC(50) values of ~7.5 and 5 μM, respectively. It dually inhibits EGFR and PKCs, resulting in a blockade of mammalian target of rapamycin signaling and cell cycle arrest in the G(1) phase, which leads to activation of apoptosis in the tumors. Hence, combinatorial inhibition of EGFR and PKCs might provide proof of concept in developing therapeutic agents for treating malignant glioma and other human cancers.     

Mesenchymal Stem Cells Modified with a Single-Chain Antibody against EGFRvIII Successfully Inhibit the Growth of Human Xenograft Malignant Glioma

Background

Glioblastoma multiforme is the most lethal brain tumor with limited therapeutic options. Antigens expressed on the surface of malignant cells are potential targets for antibody-mediated gene/drug delivery.

Principal Findings

In this study, we investigated the ability of genetically modified human mesenchymal stem cells (hMSCs) expressing a single-chain antibody (scFv) on their surface against a tumor specific antigen, EGFRvIII, to enhance the therapy of EGFRvIII expressing glioma cells in vivo. The growth of U87-EGFRvIII was specifically delayed in co-culture with hMSC-scFvEGFRvIII. A significant down-regulation was observed in the expression of pAkt in EGFRvIII expressing glioma cells upon culture with hMSC-scFvEGFRvIII vs. controls as well as in EGFRvIII expressing glioma cells from brain tumors co-injected with hMSC-scFvEGFRvIII in vivo. hMSC expressing scFvEGFRvIII also demonstrated several fold enhanced retention in EGFRvIII expressing flank and intracranial glioma xenografts vs. control hMSCs. The growth of U87-EGFRvIII flank xenografts was inhibited by 50% in the presence of hMSC-scFvEGFRvIII (p<0.05). Moreover, animals co-injected with U87-EGFRvIII and hMSC-scFvEGFRvIII intracranially showed significantly improved survival compared to animals injected with U87-EGFRvIII glioma cells alone or with control hMSCs. This survival was further improved when the same animals received an additional dosage of hMSC-scFvEGFRvIII two weeks after initial tumor implantation. Of note, EGFRvIII expressing brain tumors co-injected with hMSCs had a lower density of CD31 expressing blood vessels in comparison with control tumors, suggesting a possible role in tumor angiogenesis.

Conclusions/Significance

The results presented in this study illustrate that genetically modified MSCs may function as a novel therapeutic vehicle for malignant brain tumors.     

CD133 (Prominin) Negative Human Neural Stem Cells Are Clonogenic and Tripotent

Background

CD133 (Prominin) is widely used as a marker for the identification and isolation of neural precursor cells from normal brain or tumor tissue. However, the assumption that CD133 is expressed constitutively in neural precursor cells has not been examined.

Methodology/Principal Findings

In this study, we demonstrate that CD133 and a second marker CD15 are expressed heterogeneously in uniformly undifferentiated human neural stem (NS) cell cultures. After fractionation by flow cytometry, clonogenic tripotent cells are found in populations negative or positive for either marker. We further show that CD133 is down-regulated at the mRNA level in cells lacking CD133 immunoreactivity. Cell cycle profiling reveals that CD133 negative cells largely reside in G1/G0, while CD133 positive cells are predominantly in S, G2, or M phase. A similar pattern is apparent in mouse NS cell lines. Compared to mouse NS cells, however, human NS cell cultures harbour an increased proportion of CD133 negative cells and display a longer doubling time. This may in part reflect a sub-population of slow- or non-cycling cells amongst human NS cells because we find that around 5% of cells do not take up BrdU over a 14-day labelling period. Non-proliferating NS cells remain undifferentiated and at least some of them are capable of re-entry into the cell cycle and subsequent continuous expansion.

Conclusions

The finding that a significant fraction of clonogenic neural stem cells lack the established markers CD133 and CD15, and that some of these cells may be dormant or slow-cycling, has implications for approaches to identify and isolate neural stem cells and brain cancer stem cells. Our data also suggest the possibility that CD133 may be specifically down-regulated during G0/G1, and this should be considered when this marker is used to identify and isolate other tissue and cancer stem cells.     

Body cavity fluid can induce epithelial and mesothelial differentiation from CD34 positive peripheral blood stem cells in vitro

H. Kataoka, T. K. Kobayashi, S. Amano, E. Yamada, M. Ishida, R. Kushima and H. Okabe Body cavity fluid can induce epithelial and mesothelial differentiation from CD34 positive peripheral blood stem cells in vitro Objective: Primary culture of CD34 positive stem cells collected from human peripheral blood was performed with and without supplementation with concentrated ascitic fluid; morphological and immunocytochemical pictures of cultured cells were taken chronologically and compared. Methods: CD34‐positive stem cells collected from peripheral blood were cultured for 1, 24 and 48 hours. Concentrated ascitic fluid was added to the plates for the 24‐and 48‐hour cultures. For immunocytochemical studies, CD34, AE1/AE3, Ber‐Ep4 (EA), EMA, EGFR, CD31, CA125 and D2‐40 monoclonal antibodies were used. Results: After culture, small round cells with naked nuclei began to enlarge and to exhibit various changes in the cytoplasm and nucleus. Supplementation with concentrated body cavity fluid enhanced these changes. CD34‐positive cells with small round cell features were detected 1 hour after culture and these had no epithelial or mesothelial markers. After 24 hours, CD34‐positive cells had disappeared and cells weakly positive for EGFR, EMA, CA125 and D2‐40 were detected. Cells with strong and moderate positive reactions for EGFR, AE1/AE3, EA, EMA, D2‐40 and CA125 were detected after 48 hours. Supplementation with concentrated body cavity fluid increased the intensity and number of positive cells for these markers compared with the control group. The positive reaction, not only for the epithelial markers such as EGFR and AE1/AE3, but also for mesothelial markers such as CA125 and D2‐40, was found to be increased in small numbers of cells in direct proportion to the duration of the primary culture of the peripheral blood cells. CD31, characteristically expressed in endothelial cells, was negative in the cultured cells. Conclusion: Supplementation of peripheral blood CD34‐positive stem cells with body cavity fluid in vitro enhanced their differentiation toward cells of an epithelial or mesothelial phenotype, concomitant with loss of immunoreactivity for CD34. It is assumed that the routine cytological observation of cells obtained from body cavity fluid might cause possible cytomorphological and immunophenotypical changes due to the action of the growth factors contained in the body cavity fluid.     

Spontaneous transformation of adult mesenchymal stem cells from cynomolgus macaques in vitro

Mesenchymal stem cells (MSCs) have shown potential clinical utility in cell therapy and tissue engineering, due to their ability to proliferate as well as to differentiate into multiple lineages, including osteogenic, adipogenic, and chondrogenic specifications. Therefore, it is crucial to assess the safety of MSCs while extensive expansion ex vivo is a prerequisite to obtain the cell numbers for cell transplantation. Here we show that MSCs derived from adult cynomolgus monkey can undergo spontaneous transformation following in vitro culture. In comparison with MSCs, the spontaneously transformed mesenchymal cells (TMCs) display significantly different growth pattern and morphology, reminiscent of the characteristics of tumor cells. Importantly, TMCs are highly tumorigenic, causing subcutaneous tumors when injected into NOD/SCID mice. Moreover, no multiple differentiation potential of TMCs is observed in vitro or in vivo, suggesting that spontaneously transformed adult stem cells may not necessarily turn into cancer stem cells. These data indicate a direct transformation of cynomolgus monkey MSCs into tumor cells following long-term expansion in vitro. The spontaneous transformation of the cultured cynomolgus monkey MSCs may have important implications for ongoing clinical trials and for models of oncogenesis, thus warranting a more strict assessment of MSCs prior to cell therapy.     

Cellular antitumor immune response to a branched lysine multiple antigenic peptide containing epitopes of a common tumor-specific antigen in a rat glioma model

Human malignant gliomas contain epidermal growth factor receptor (EGFR) gene mutations that encode tumor-associated antigens (TAAs) that can be targeted using immunological techniques. One EGFR mutant gene (EGFRvIII) encodes a protein with an epitope that is not found in normal tissues. A number of studies have focused on this unique epitope as a potential target for tumor vaccines. In the present study, we examined the cellular immune effects of a peptide containing multiple copies of the unique EGFRvIII epitope linked together by way of a lysine bridge. Fischer rats were vaccinated with an EGFRvIII multiple antigenic peptide (MAP). While vaccination produced a humoral immune response, anti-MAP antibody production was not accompanied by expression of the Th2 response cytokine IL-4. In MAP/GM-CSF vaccinated animals, a cellular immune response was detected in association with the appearance of CD4⁺ and CD8⁺ T cells at the tumor site. Splenocytes and CD8⁺ T cells from vaccinated rats produced the Th1 cytokine IFN- in vitro in response to stimulation by rat glioma cells expressing EGFRvIII, but not by those expressing wild-type EGFR. MAP vaccine also induced a specific lytic antitumor CTL immune response against F98 glioma cells expressing EGFRvIII, but not against F98 cells expressing either wild-type EGFR or no receptor. The in vivo growth of F98_EGFRvIII cells was attenuated in vaccinated rats; whereas, growth of F98_EGFR cells was not. The median survival of vaccinated rats was increased 72% over that of unvaccinated controls challenged with intracerebral F98_EGFRvIII tumor implants. Therefore, MAP vaccination produced a predominantly cellular antitumor immune response directed against F98 gliomas expressing the EGFRvIII target antigen. The potent immunosuppressive effects of F98 glioma cells mimic the human disease and make this particular tumor model useful for studying immunotherapeutic approaches to malignant gliomas.     

Characterization of CD133+ hepatocellular carcinoma cells as cancer stem/progenitor cells

The CD133 antigen, identified as a hematopoietic stem cell marker, appears in various human embryonic epithelia including the neural tube, gut, and kidney. We herein investigated whether CD133(+) cells isolated from human hepatocellular carcinoma cell lines possess cancer stem/progenitor cell-like properties. Among the three cell lines studied, the CD133 antigen was found to be expressed only on the surface of Huh-7 cells. CD133(+) cells from Huh-7 performed a higher in vitro proliferative potential and lower mRNA expressions of mature hepatocyte markers, glutamine synthetase and cytochrome P450 3A4, than CD133(-) population of Huh-7 cells. When either CD133(+) or CD133(-) cells were subcutaneously injected into SCID mice, CD133(+) cells formed tumors, whereas CD133(-) cells induced either a very small number of tumors or none at all. Taken together, the identification of CD133(+) cells could thus be a potentially powerful tool to investigate the tumorigenic process in the hepatoma system and to also develop effective therapies targeted against hepatocellular carcinoma.     

A new role of thrombopoietin enhancing ex vivo expansion of endothelial precursor cells derived from AC133-positive cells

We previously reported that CD31(bright) cells, which were sorted from cultured AC133(+) cells of adult peripheral blood cells, differentiated more efficiently into endothelial cells than CD31(+) cells or CD31(-) cells, suggesting that CD31(bright) cells may be endothelial precursor cells. In this study, we found that CD31(bright) cells have a strong ability to release cytokines. The mixture of vascular endothelial growth factor (VEGF), thrombopoietin (TPO), and stem cell factor stimulated ex vivo expansion of the total cell number from cultured AC133(+) cells of adult peripheral blood cells and cord blood cells, resulting in incrementation of the adhesion cells, in which endothelial nitric oxide synthase and kinase insert domain-containing receptor were positive. Moreover, the mixture of VEGF and TPO increased the CD31(bright) cell population when compared with VEGF alone or the mixture of VEGF and stem cell factor. These data suggest that TPO is an important growth factor that can promote endothelial precursor cells expansion ex vivo.     

CD31 (PECAM-1)-bright cells derived from AC133-positive cells in human peripheral blood as endothelial-precursor cells

To clarify the process of endothelial differentiation, we isolated AC133(+) cells and induced the in vitro differentiation of these cells into endothelial cells. AC133(+) cells efficiently differentiated into endothelial cells when the cells were cultured on fibronectin-coated dishes in the presence of vascular endothelial growth factor. Time-course analysis of the alteration of endothelial markers on cultured AC133(+) cells revealed that the expression of CD31 (PECAM-1) on AC133(+) cells was the earliest marker among all of the tested markers. Based on the hypothesis that CD31 is an early indicator during the endothelial differentiation, we examined the relationship between CD31 expression and the ability to differentiate into endothelial cells in cells derived from AC133(+) cells. CD31-bright cells, which were sorted from cultured AC133(+) cells, differentiated more efficiently into endothelial cells than had CD31-positive or CD31-negative cells, suggesting that CD31-bright cells may be precursor cells for endothelial cells. In the present study, we identified CD31(+) cells derived from cultured AC133(+) cells that are able to differentiate to endothelial cells as precursor cells.     

Isolation of EpCAM+/CD133− hepatic progenitor cells

Progenitor cell-derived hepatocytes are critical for hepatocyte replenishment. Therefore, we established a line of human hepatic progenitor (HNK1) cells and determined their biological characteristics for experimental and therapeutic applications. HNK1 cells, isolated from human noncirrhotic liver samples with septal fibrosis, showed high expression of the hepatic progenitor cell (HPC) markers EpCAM, CK7, CK19, alpha-fetoprotein (AFP), CD90 (Thy1), and EFNA1. Expression of CD133 was very low. Ductular reactions at the periphery of cirrhotic nodules were immunohistochemically positive for these HPC markers, including EFNA1. Sodium butyrate, a differentiation inducer, induced hepatocyte-like morphological changes in HNK1 cells. It resulted in down-regulation of the hepatic progenitor cell markers EpCAM, CK7, CK19, AFP, and EFNA1 and up-regulation of mature hepatocyte markers, including albumin, CK8, and CK18. Furthermore, sodium butyrate treatment and a serial passage of HNK1 cells resulted in enhanced albumin secretion, ureagenesis, and CYP enzyme activity, all of which are indicators of differentiation in hepatocytes. However, HNK1 cells at passage 50 did not exhibit anchorage-independent growth capability and caused no tumors in immunodeficient mice, suggesting that they had no spontaneous malignant transformation ability. From this evidence, HNK1 cells were found to be EpCAM⁺/CD133^- hepatic progenitor cells without spontaneous malignant transformation ability. We therefore conclude that HNK1 cells could be useful for experimental and therapeutic applications.     

Surface antigenic profiling of stem cells from human omentum fat in comparison with subcutaneous fat and bone marrow

Omentum fat derived stem cells have emerged as an alternative and accessible therapeutic tool in recent years in contrast to the existing persuasive sources of stem cells, bone marrow and subcutaneous adipose tissue. However, there has been a scanty citation on human omentum fat derived stem cells. Furthermore, identification of specific cell surface markers among aforesaid sources is still controversial. In lieu of this existing perplexity, the current research work aims at signifying omentum fat as a ground-breaking source of stem cells by surface antigenic profiling of stem cell population. In this study, we examined and compared the profiling of cell surface antigenic expressions of hematopoietic stem cells, mesenchymal stem cells, cell adhesion molecules and other unique markers such as ABCG2, ALDH and CD 117 in whole cell population of human omentum fat, subcutaneous fat and bone marrow. The phenotypic characterization through flowcytometry revealed the positive expressions of CD 34, CD 45, CD 133, HLADR, CD 90, CD 105, CD 73, CD 29, CD 13, CD 44, CD 54, CD 31, ALDH and CD 117 in all sources. The similarities between the phenotypic expressions of omentum fat derived stem cells to that of subcutaneous fat and bone marrow substantiates that identification of ultimate source for curative therapeutics is arduous to assess. Nevertheless, these results support the potential therapeutic application of omentum fat derived stem cells.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-012-9427-4) contains supplementary material, which is available to authorized users.     

Doxycycline Induces Apoptosis and Inhibits Proliferation and Invasion of Human Cervical Carcinoma Stem Cells

BackgroundCancer stem cells (CSCs) are proposed to be responsible for high recurrence rate in cervical carcinoma. Reagents that can suppress the proliferation and differentiation of CSCs would provide new opportunities to fight against tumor recurrence. Doxycycline has been reported as a potential anti-cancer compound. However, few studies investigated its inhibitory effect against cervical cancer stem cells.MethodsHeLa cells were cultured in cancer stem cell conditional media in a poly-hema-treated dish. In this non-adhesive culture system, HeLa cells were treated with cisplatin until some cells survived and formed spheroids, which were then collected and injected into the immunodeficient mice. Cisplatin was administered every three days for five times. The tumor xenografts with CSC enrichment were cultured in cancer stem cell specific medium again to form tumorsphere, which we called HeLa-CSCs. Expression of cancer stem cell markers in HeLa-CSCs was measured by flow cytometry and qPCR. HeLa-CSCs were then treated with doxycycline. Proliferation and differentiation rates were determined by the size of spheres formed in vitro and tumor formed in vivo.ResultsWe developed a new strategy to selectively enrich CSCs from human cervical carcinoma cell line HeLa, and these HeLa-CSCs are CD133+/CD49f+ cell populations with significantly enhanced expression of stem cell markers. When these HeLa-CSCs were treated with doxycycline, the colony formation, proliferation, migration and invasion, and differentiation were all suppressed. Meanwhile, stem cell markers SOX-2, OCT-4, NANOG, NOTCH and BMI-1 decreased in doxycycline treated cells, so as the surface markers CD133 and CD49f. Furthermore, proliferation markers Ki67 and PCNA were also decreased by doxycycline treatment in the in vivo xenograft mouse model.ConclusionsCancer stem cells are enriched from sphere-forming and chemoresistant HeLa-derived tumor xenografts in immunodeficient mice. Doxycycline inhibits proliferation, invasion, and differentiation, and also induces apoptosis of these HeLa-CSCs in vitro and in vivo.     

Gene analysis and dynamics of tumor stem cells in human glioblastoma cells after radiation

Glioblastoma is the most malignant central nervous system tumor. Patients with glioblastoma are treated with a combination of surgery, radiotherapy and chemotherapy; however, this effect is not satisfactory with regard to the prognosis. It is reported that the tumor stem cells affect recurrence, and radio- and chemotherapy resistance of the tumor, and that these cells play an important role in tumorigenesis and tumor progression. Using human glioblastoma cell lines (T98G and A172), irradiated (0, 30, 60 Gy) glioblastoma cells were prepared under the same conditions as clinical therapy. We analyzed cell proliferation rate, side population analysis by fluorescence-activated cell sorting and isolation of CD133⁺ cells, and performed genetic analysis (human stem cells) on these cells. We also investigated the difference in gene expression in the cells after radiation. The stem cell-related genes were highly expressed in the CD133⁺ cells compared with the CD133^? cells, suggesting that the cancer stem cells may be located in these CD133⁺ cells. In the T98G cell line, the cell proliferation rate of 30-Gy irradiated cells was higher than those of non-irradiated cells and 60-Gy irradiated cells. Stem cell-related genes were highly expressed in 30-Gy irradiated CD133⁺ T98G cells. In conclusion, we suggest that CD133⁺ cells may strongly affect tumor proliferation and the resistance against radiation therapy.     

Telomerase-immortalized non-malignant human prostate epithelial cells retain the properties of multipotent stem cells

Understanding prostate stem cells may provide insight into the origin of prostate cancer. Primary cells have been cultured from human prostate tissue but they usually survive only 15-20 population doublings before undergoing senescence. We report here that RC-170N/h/clone 7 cells, a clonal cell line from hTERT-immortalized primary non-malignant tissue-derived human prostate epithelial cell line (RC170N/h), retain multipotent stem cell properties. The RC-170N/h/clone 7 cells expressed a human embryonic stem cell marker, Oct-4, and potential prostate epithelial stem cell markers, CD133, integrin alpha2beta1(hi) and CD44. The RC-170N/h/clone 7 cells proliferated in KGM and Dulbecco's Modified Eagle Medium with 10% fetal bovine serum and 5 microg/ml insulin (DMEM+10% FBS+Ins.) medium, and differentiated into epithelial stem cells that expressed epithelial cell markers, including CK5/14, CD44, p63 and cytokeratin 18 (CK18); as well as the mesenchymal cell markers, vimentin, desmin; the neuron and neuroendocrine cell marker, chromogranin A. Furthermore the RC170 N/h/clone 7 cells differentiated into multi tissues when transplanted into the sub-renal capsule and subcutaneously of NOD-SCID mice. The results indicate that RC170N/h/clone 7 cells retain the properties of multipotent stem cells and will be useful as a novel cell model for studying the mechanisms of human prostate stem cell differentiation and transformation.     

Cloned murine non-malignant, spontaneously transformed and chemical tumour-derived cell lines related to the type 2 pneumocyte

NAL1A is a murine type 2 pneumocyte-related cell line cultured from normal BALB/c adult mouse lung. In vitro spontaneous transformation of 3 out of 7 clones of NAL1A has led to the isolation and establishment in continuous cell culture of sibling-related non-neoplastic (NAL1A) and spontaneously arising neoplastic (NAL1As) cell strains. NAL1As cells exhibited a similar phenotype to cloned NUL1 cells cultured from urethane-induced mouse lung adenomas. All NAL1As and NUL1 clones grew vigorously in 0.3% agar and formed invasive, poorly differentiated carcinomas following subcutaneous inoculation into immunesuppressed mice. Several subcutaneous nodules metastasised preferentially to the lung. All spontaneous and chemically-derived malignant clones were less differentiated than the non-malignant clones as assessed by staining with a type 2 pneumocyte-specific polyclonal antiserum. The clones described in this report form a useful model in the study of spontaneous and chemically-induced neoplastic transformation in mouse epithelial lung cells.