Immortalized mouse fetal liver stromal cells support growth and maintenance of human embryonic stem cells

Human embryonic stem cells (hESCs) are usually maintained in an undifferentiated state by co-culture with feeder cells. The feeder cells are important for the growth of hESCs. A novel spontaneously immortalizated mouse fetal liver stromal cell line, named KM3, was isolated from a?13.5?day mouse fetal liver. In this study, we examined whether KM3 cells could be used as feeders to support the growth of hESCs. hESCs cultured on KM3 cells showed a similar proliferation rate and characteristics to mouse embryonic fibroblasts?(MEFs) after prolonged culture, including morphology, unlimited and undifferentiated proliferative ability, maintenance of normal karyotypes, formation of embryoid bodies in?vitro and typically immature teratomas in?vivo. Our results indicate that the immortalized KM3 cell line has the potential to support the growth and maintenance of hESCs. The cell line may be used for the large-scale expansion of hESCs in a low-cost and less labor-intensive manner.     

Differentiation of human embryonic stem cells into immunostimulatory dendritic cells under feeder-free culture conditions

PURPOSE: The objective of this study was to develop a scalable and broadly applicable active immunotherapy approach against cancer, circumventing the limitations typically encountered with autologous vaccination strategies. We hypothesized that human embryonic stem cells (hESC) can serve as a virtually unlimited source for generating dendritic cells (DC) with potent antigen-presenting function. Here, we investigated the developmental processes and requirements for generating large numbers of mature, antigen-presenting DC from pluripotent hESC. EXPERIMENTAL DESIGN: A feeder cell-free culture system was developed to differentiate hESC into mature DC sequentially through hematopoietic and myeloid precursor stages. RESULTS: Using this method, we were able to yield large numbers of mature immunostimulatory DC from hESC to enable clinical investigation. Upon activation, the hESC-derived DC secreted interleukin-12p70, migrated in response to MIP-3beta, and exhibited allostimulatory capacity. Most importantly, antigen-loaded, hESC-derived DC were capable of stimulating potent antigen-specific CD8(+) T-cell responses in an HLA class I-matched semiallogeneic assay system. Moreover, HLA class II-mismatched hESC-derived DC induced a potent Th1-type cytokine response without expanding FOXP3(+) regulatory T cells in vitro. CONCLUSIONS: These data suggest the development of a novel active immunotherapy platform to stimulate potent T-cell immunity in patients with intractable diseases, such as cancer or viral infection.     

Deficiency of Retinoblastoma gene in mouse embryonic stem cells leads to genetic instability

Genetic instability has been recognized as a hallmark of human cancers. Retinoblastoma (Rb) tumor suppressor protein has an essential role in modulating cell cycle progression. However, there is no direct evidence supporting its role in maintaining genetic stability. Here, we developed a sensitive method to examine the level of chromosome instability by using retrovirus carrying both positive and negative selectable markers that integrated randomly into individual chromosomes, and the frequency of loss of this selectable chromosomal marker (LOM) in normal mammalian cells was measured. Our results showed that normal mouse embryonic stem (ES) cells had a very low frequency of LOMs, which was less than 10(-8)/cell/generation. In Rb-/- mouse ES cells, the frequency was increased to approximately 10(-5)/cell/generation, whereas in Rb+/- ES cells, the frequency was approximately 10(-7)/cell/generation. LOMs was mediated mainly through chromosomal mechanisms and not through point mutations. These results, therefore, revealed that Rb, with a haploinsufficiency, plays a critical role in the maintenance of chromosome stability. The mystery of why Rb heterozygous carriers have early-onset tumor formation with high penetrance can be, at least, partially explained by this novel activity.     

LIGHT induces differentiation of mouse embryonic stem cells associated with activation of ERK5

LT-related inducible ligand that competes for glycoprotein D binding to herpesvirus entry mediator on T cells (LIGHT) is a recently cloned type II transmembrane protein belonging to the TNF family that was originally identified as a weak inducer of apoptosis. This cytokine has been extensively defined in its role on T-cell regulation and dendritic cell maturation. However, whether this cytokine regulates stem cell proliferation and/or differentiation remains unknown. In this study, we transduced exogenous LIGHT into embryonic stem cells (ES cells) and found it induced their differentiation. The expression of phospho-STAT3, Nanog and Oct-4 was reduced in LIGHT-transduced ES cells compared with wild-type ES cells. LIGHT-transduced ES cells exhibit a low level of SSEA-1 surface antigen and alkaline phosphatase staining compared with wild-type cells. Introduction of LIGHT into ES cells results in the dephosphorylation of MKP-3 and activation of extracellular signal-regulated kinase (ERK)5. When ERK5 was inhibited by the specific inhibitor PD184352 or knocked down by ERK5 siRNA, reduction of Oct-4 and SSEA-1 expression was rescued. We conclude that LIGHT overrides Leukemia inhibitory factor to induce ES cell differentiation associated with activation of ERK5.     

Gene expression profiling of mouse teratocarcinomas uncovers epigenetic changes associated with the transformation of mouse embryonic stem cells

The molecular mechanisms of the development of teratocarcinomas from stem cells are largely unknown. To determine which genes are associated with the transformation of these cells, we have performed oligonucleotide microarray analysis, using Affymetrix U74A GeneChips, on both cell cultures and tumors in nude mice. We identified 68 genes that significantly differed in expression between the ES cell culture and the teratocarcinoma cell line, SCC-PSA1, and 51 genes with statistically different expression patterns between the ES cell tumors and the teratocarcinomas (P < .00005). We found that there were 20 genes that had common expression patterns in both groups. We also examined the role of the transition from in vitro to in vivo by comparing ES cell culture to ES cell tumor, and teratocarcinoma cell line to teratocarcinomas. We identified 22 genes that were upregulated in the ES cell tumors and 42 that had a decreased expression in the tumor (P < .0001). In comparing SCC-PSA1 to its tumor, we identified 34 upregulated genes and 25 downregulated genes (P < .001). There were only 10 genes in common from these two lists. GenMapp search revealed that several pathways, especially the cell cycle pathway, are actively involved in the induction of teratocarcinomas. Our results indicate that many key development genes may play a key role in the transformation of ES cells into teratocarcinoma cells.     

Msh2 deficiency does not contribute to cisplatin resistance in mouse embryonic stem cells

Several reports have suggested that a defect in the DNA mismatch repair (MMR) system not only causes resistance to methylating agents but also confers low-level resistance to the chemotherapeutic drug cisplatin. Here we report that in a clonogenic assay, mouse embryonic stem (ES) cells deficient for the MMR protein MSH2 respond similarly as wild-type cells to cisplatin. Furthermore, restoring MSH2 expression in a cisplatin-resistant subclone selected from an Msh2(-/-) cell population did not sensitize cells to cisplatin. To ascertain that our observations were not the result of a mutation in the Msh2(-/-) cells that obscured the contribution of a defective MMR machinery to cisplatin resistance, we made use of the Cre-lox system to create a cell line in which the Msh2 gene can be conditionally inactivated. However, while de novo inactivation of Msh2 rendered cells tolerant to the methylating drug N-methyl-N'-nitro-N-nitrosoguanidine as expected, it did not alter the sensitivity to cisplatin. In addition, we were not able to derive cisplatin-resistant subclones from this freshly generated MMR-deficient cell line. Thus, in ES cells we did not find evidence for direct involvement of MMR deficiency in cisplatin resistance.     

Influence of mouse uterus on the metastatic patterns of tumour cells

Metastatic patterns of fibrosarcoma (FS) and Lewis lung carcinoma (LLC) cells transplanted into mouse uteri of various reproductive stages, were investigated. Tumour cells were infused non-surgically into the lumen of 3-day post coitum pregnant uterus or into non-pregnant uterus of known estrus stage. The fate of these tumour cells was studied histologically on days 2, 5 and 10 post treatment. No significant difference in the metastatic patterns of the FS or the LLC cells was seen between the non-pregnant uteri of various estrus stages. FS cells in few cases of non-pregnant uteri displayed the tendency to migrate and grow outside the myometrium without colonizing in the endometrium, but in pregnant uteri they colonized within the endometrium. LLC cells in the non-pregnant uteri promptly metastasized to distant organs like liver and lung; but those in the pregnant uteri rarely metastasized to other organs. These observations imply that the metastatic patterns of uterine tumour might depend on both the physiological state of the uterus and the tumour cell type.     

Activation of Akt signaling is sufficient to maintain pluripotency in mouse and primate embryonic stem cells

Embryonic stem (ES) cells can self-renew indefinitely without losing their differentiation ability to any cell types. Phosphoinositide-3 kinase (PI3K)/Akt signaling plays a pivotal role in various stem cell systems, including the formation of embryonic germ (EG) cells from primordial germ cells and self-renewal of neural stem cells. Here, we show that myristoylated, active form of Akt (myr-Akt) maintained the undifferentiated phenotypes in mouse ES cells without the addition of leukemia inhibitory factor (LIF). The effects of myr-Akt were reversible, because LIF dependence and pluripotent differentiation activity were restored by the deletion of myr-Akt. In addition, myr-Akt-Mer fusion protein, whose enzymatic activity is controlled by 4-hydroxy-tamoxifen, also maintained the pluripotency of not only mouse but also cynomolgus monkey ES cells. These results clearly demonstrate that Akt signaling sufficiently maintains pluripotency in mouse and primate ES cells, and support the notion that PI3K/Akt signaling axis regulates 'stemness' in a broad spectrum of stem cell systems.     

Mechanisms of self-renewal in human embryonic stem cells

Embryonic stem cells (ESCs) are the pluripotent cell population derived from the inner cell mass of pre-implantation embryos and are characterised by prolonged self-renewal and the potential to differentiate into cells representing all three germ layers both in vitro and in vivo. Preservation of the undifferentiated status of the ESC population requires the maintenance of self-renewal whilst inhibiting differentiation and regulating senescence and apoptosis. In this review, we discuss the intrinsic and extrinsic factors associated with self-renewal process, together with possible signalling pathway interactions and mechanisms of regulation.     

Resveratrol protects mouse embryonic stem cells from ionizing radiation by accelerating recovery from DNA strand breakage

Resveratrol has elicited many provocative anticancer effects in laboratory animals and cultured cells, including reduced levels of oxidative DNA damage, inhibition of tumor initiation and progression and induction of apoptosis in tumor cells. Use of resveratrol as a cancer-preventive agent in humans will require that its anticancer effects not be accompanied by damage to normal tissue stem or progenitor cells. In mouse embryonic stem cells (mESC) or early mouse embryos exposed to ethanol, resveratrol has been shown to suppress apoptosis and promote survival. However, in cells exposed to genotoxic stress, survival may come at the expense of genome stability. To learn whether resveratrol can protect stem cells from DNA damage and to study its effects on genomic integrity, we exposed mESC pretreated with resveratrol to ionizing radiation (IR). Forty-eight hours pretreatment with a comparatively low concentration of resveratrol (10 μM) improved survival of mESC >2-fold after exposure to 5 Gy of X-rays. Cells pretreated with resveratrol sustained the same levels of reactive oxygen species and DNA strand breakage after IR as mock-treated controls, but repaired DNA damage more rapidly and resumed cell division sooner. Frequencies of IR-induced mutation at a chromosomal reporter locus were not increased in cells pretreated with resveratrol as compared with controls, indicating that resveratrol can improve viability in mESC after DNA damage without compromising genomic integrity.     

Effects of bleomycin on mouse bone-marrow stem cells.

The mouse hematopoietic stem-cell population was tested by the spleen colony technique for effects of the antineoplastic agent bleomycin (BLM). The time response of normal bone marrow was investigated by a single dose of BLM (400 mg/kg) between 0 and 72 hours. The dose response was studied over a wide range of doses (from 40 to 1,600 mg/kg) at a 4-hour exposure. Additional experiments concerned 1) the fraction of colony-forming units in the S phase after BLM administration (by means of pulse hydroxyurea treatment), 2) the response of bone marrow stimulated by endotoxin, and 3) the effects of split-dose treatments. The relatively low toxicity of BLM on both the differentiated and stem-cell populations of unstimulated bone marrow was confirmed and detailed. This drug exhibited peculiar, proliferation-dependent cell inactivation kinetics. Furthermore, BLM induced parasynchronous behavior in the unstimulated stem-cell population. The various aspects of BLM action are discussed with regard to its use in cancer chemotherapy.     

Neoplastic human embryonic stem cells as a model of radiation resistance of human cancer stem cells

Studies have implicated that a small sub-population of cells within a tumour, termed cancer stem cells (CSCs), have an enhanced capacity for tumour formation in multiple cancers and may be responsible for recurrence of the disease after treatment, including radiation. Although comparisons have been made between CSCs and bulk-tumour, the more important comparison with respect to therapy is between tumour-sustaining CSC versus normal stem cells that maintain the healthy tissue. However, the absence of normal known counterparts for many CSCs has made it difficult to compare the radiation responses of CSCs with the normal stem cells required for post-radiotherapy tissue regeneration and the maintenance of tissue homeostasis. Here we demonstrate that transformed human embryonic stem cells (t-hESCs), showing features of neoplastic progression produce tumours resistant to radiation relative to their normal counterpart upon injection into immune compromised mice. We reveal that t-hESCs have a reduced capacity for radiation induced cell death via apoptosis and exhibit altered cell cycle arrest relative to hESCs in vitro. t-hESCs have an increased expression of BclXL in comparison to their normal counterparts and re-sensitization of t-hESCs to radiation upon addition of BH3-only mimetic ABT737, suggesting that overexpression of BclXL underpins t-hESC radiation insensitivity. Using this novel discovery platform to investigate radiation resistance in human CSCs, our study indicates that chemotherapy targeting Bcl2-family members may prove to be an adjuvant to radiotherapy capable of targeting CSCs.     

Effect of age and sex on lung-colony-forming efficiency of injected mouse tumour cells.

The i.v. injection of a specified number of cells of either an Ehrlich ascites tumour (ELD) or spontaneous mouse mammary adenocarcinomas (MA) into C3H mice yielded a number of lung colonies which varied significantly with the age or sex of recipient mice. The yield was higher in mice of 71 weeks than in those of 15 weeks, except for MA cells injected into females, when the yield was higher in the younger mice. Sex did not influence very significantly the yield of colonies from ELD cells; in the case of MA cells the direction of sex differences depended on age. A difference in the effect of pre-immunization with age was not observed.     

小白菊内酯体内杀伤小鼠乳腺癌肿瘤干细胞

目的： 探讨小白菊内酯（parthenolide,PTL）对小鼠乳腺癌肿瘤干细胞（cancer stem cell,CSC）的杀伤作用,为临床应用PTL治疗乳腺癌提供实验依据。 方法： 采用5-氟尿嘧啶（5-fluorouracil, 5-FU）化疗法制备富含CSC的小鼠4T1细胞乳腺癌模型,随机分为对照组、5-FU组、PTL组。4周后脱颈处死小鼠,检测各组小鼠肿瘤的体积和重量,流式细胞术检测小鼠肿瘤组织中CD44+CD24-/low细胞比例,Hoechst33342染色法检测侧群（side population,SP）细胞的比例,免疫组化法检测CD55和乙醛脱氢酶1（aldehyde dehydrogenase1,ALDH1）蛋白的表达,倒置显微镜观察乳腺癌细胞微球体的形成。 结果： 成功制备富含CSC的小鼠乳腺癌细胞移植瘤模型,PTL可下调小鼠肿瘤组织中CD44+CD24-/low细胞的比例\[（42.5±3.7）% vs （68.7±32）%,P<0.05\],有效降低荷瘤小鼠肿瘤组织中SP细胞的比例\[（39.2±1.8）% vs （61.3±2.6）%,P<0.05\],下调小鼠移植瘤组织中CD55和ALDH1蛋白的表达\[（18.9±1.5）% vs （30.1±1.3）%,（8.1±2.3）% vs （18.0±1.4）%;均P<0.05\],抑制小鼠肿瘤细胞在无血清培养条件下形成微球体,并可抑制小鼠移植瘤的体积和重量\[（0.625±0.159）cm3 vs （1.715±0184）cm3,（1.467±0.373）g vs （3.367±0.398）g;均P<0.05\]。 结论： PTL在荷瘤小鼠体内可以明显降低肿瘤组织CSC含量,提示PTL可用来靶向杀伤乳腺癌CSC。     

Development of a rabbit monoclonal antibody group against Smads and immunocytochemical study of human and mouse embryonic stem cells

We report here the development of a group of rabbit monoclonal antibodies against Smad1, Smad2, Smad3, and Smad5, and the immunocytochemistry (ICC) staining of human embryonic stem cells (hESC) and mouse embryonic stem cells (mESC). Eight New Zealand rabbits were immunized with synthesized peptides linked to KLH, and splenocytes from these rabbits were fused with rabbit immortal B cell 240E-W2. Resulting hybridomas producing anti-Smad monoclonal antibodies were screened by enzyme-linked immunosorbent assay (ELISA) with BSA-linked peptides. Clones were chosen for antibody production based on their activities in Western blotting and on paraffin-embedded human tissues, and the capacity of the antibodies in immunocytochemistry was demonstrated. Using these antibodies, we performed ICC staining on routinely cultured human and mouse embryonic stem cells, and showed that both cell types strongly express these genes. We propose that both hESCs and mESCs have the ability to transduce signals from both BMPs and TGF-b/Activin.     

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.     

Toxic effect of polyriboinosinic-polyribocytidylic acid on mouse hemopoietic stem cells

Within a few hours following a single intraperitoneal injection of polyriboinosinic-polyribocytidylic acid (poly I:poly C) into C3H/He mice, numbers of bone-marrow and spleen-colony-forming cells are significantly decreased. The dose-response curve obtained shows an exponential decrease of the surviving CFU with increasing amounts of poly I:poly C, up to 200 μg; with higher doses of poly I:poly C, no further decrease of CFU is observed. This suggests that poly I:poly C kills colony-forming cells in replicating phase, and is devoid of activity on cells in resting phase. The lack of additive toxic effects observed when poly I:poly C and vinblastine sulfate are administred simultaneously is consistent with this hypothesis. The cytotoxic effect of the polynucleotide is related to its double-strandedness and is not mediated by interferon present in the serum.     

Growth control of heterologous tissue culture cells in the congenitally athymic nude mouse.

A variety of heterologous mammalian cells were inoculated into nude mice and scored for tumorigenicity. The cells tested were from primary cell cultures, established cell lines of neoplastic origin, established cell lines of nontumor origin, and primary cell cultures transformed by oncogenic viruses. Regardless of the animal species of origin, every cell line that was tumorigenic in some other animal host and every cell line of neoplastic origin was tumorigenic in nude mice. Several tissue culture cells lines capable of indefinite growth in vitro failed to form tumors in nude mice, and the basis of this growth suppression was investigated. The findings suggest that the failure of an established cell line to form tumors in nude mice is an authentic response to host-mediated growth-regulatory signals.